Previous in vivo study demonstrated that beta gamma-CAT, a newly identified non-lens beta gamma-crystallin and trefoil factor complex from frog Bombina maxima skin secretions, possessed potent lethal toxicity on mammals resulted from hypotension and cardiorespiratory arrest. However, the mechanism of cardiac dysfunction induced by the protein is unknown. Here, we report that beta gamma-CAT, with dosages of 0.8-3.0 nM, elicited an acute negative inotropic effect in isolated rabbit heart Langensdorff preparations, which mimicked acute heart failure. In addition, the effect of beta gamma-CAT on the hearts was mediated by endothelium-dependent coronary vasoconstriction (P < 0.01, compared between endothelium-intact and removal hearts). After beta gamma-CAT (3.0 nM) treatment, the positive signal of tumor necrosis factor-alpha (TNF-alpha) was detected mainly around the endothelial cell layer as detected by in situ indirect immunofluorescence, indicating that the release of TNF-a occurred. At the same time, a rapid TNF-a release was detected in primary cultured rabbit endocardial endothelial cells (REECs) treated with fly-CAT. After addition of beta gamma-CAT (3.0 nM) for 10 min and 30 min, the TNIF-alpha levels were increased to 57.33 +/- 3.22 pg/ml and 60.00 +/- 5.35 pg/ml (P < 0.05, compared with the control values of 21.67 +/- 3.45 pg/ml and 33.70 +/- 6.24 pg/.ml, respectively). At high concentrations, beta gamma-CAT interfered with the cell viability of REECs (CC50 about 25 nM). Taken together, beta gamma-CAT was able to induce acute myocardial depression and the toxic effect might be partially explained by the release of TNF-alpha. The finding provides new information to understand the patho-physiological roles of non-lens beta gamma-crystallins and trefoil factors. (C) 2008 Elsevier Ltd. All rights reserved.