βγ-CAT, a non-lens βγ-crystallin and trefoil factor complex from amphibian skin secretions, caused endothelium-dependent myocardial depression in isolated rabbit hearts

Previous in vivo study demonstrated that βγ-CAT, a newly identified non-lens βγ-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 βγ-CAT, with dosages of 0.8–3.0 nM, elicited an acute negative inotropic effect in isolated rabbit heart Langendorff preparations, which mimicked acute heart failure. In addition, the effect of βγ-CAT on the hearts was mediated by endothelium-dependent coronary vasoconstriction (P < 0.01, compared between endothelium-intact and removal hearts). After βγ-CAT (3.0 nM) treatment, the positive signal of tumor necrosis factor-α (TNF-α) was detected mainly around the endothelial cell layer as detected by in situ indirect immunofluorescence, indicating that the release of TNF-α occurred. At the same time, a rapid TNF-α release was detected in primary cultured rabbit endocardial endothelial cells (REECs) treated with βγ-CAT. After addition of βγ-CAT (3.0 nM) for 10 min and 30 min, the TNF-α 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, βγ-CAT interfered with the cell viability of REECs (CC50 about 25 nM). Taken together, βγ-CAT was able to induce acute myocardial depression and the toxic effect might be partially explained by the release of TNF-α. The finding provides new information to understand the patho-physiological roles of non-lens βγ-crystallins and trefoil factors.