Study of the effect of A-769662, a direct AMPK activator, on myocardial function and TLR-4 activity in isolated rat hearts under LPS perfusion

Abstract

Introduction: TLR-4 activates a number of inflammatory signaling pathways through MyD88. The aim of this study was to investigate the effect of AMPK activation on the inflammatory signaling induced by TLR-4 in the heart. It is considered that AMPK could be involved in anti-inflammatory signaling. Aims and methods: To assess the possible presence of TLR-4 as a local immune response in the heart tissue, the effect of LPS (TLR-4 ligand) on cardiac function and TLR-4 expression were examined using langendorff method. To evaluate the suppressive effect of AMPK on the myocardial TLR-4 activity the effects of A-769662, a direct AMPK activator, and metformin were studied on LPS (i.p.) induced myocardial dysfunction in vivo models in mice and Wistar rats. In this theses in addition to histological and biochemical analysis, we used real-time PCR, western blotting, and ELISA techniques. Results: In isolated rat heart, perfusion of LPS at 1 µg/mL for 180 minutes increased CPP by 37.26% compare to the control value. LVDP and both dP/dtmax and dP/dtmin was depressed to 41.5%, 24.5% and 34.4%, respectively. The suppression was associated with a significant increase in TLR-4 expression (p<0.01), MyD88 activity (p<0.01), and TNF-α (p<0.05) concentration in the heart tissue. A-769662 attenuated the LPS-induced acute inflammation as evidenced by a marked reduction in MPO activity in heart and lung (p<0.01), in serum level of TNF-α (p<0.01), and in peripheral neutrophils count (p<0.001). Furthermore, A-769662 along with LPS downregulated the expression of MyD88 (p<0.01) and enhanced AMPK phosphorylation (p<0.01) in the heart tissue Both A-769662 and metformin alleviated the effect of LPS on TLR-4 expression (p<0.01 for A-769662) and peripheral neutrophil count (p<0.01, for both). However, antagonizing AMPK activity by compound C reversed these protective effects (for TLR-4: p<0.001 and p<0.05, respectively and for peripheral neutrophil count: p<0.05). Besides, metformin suppressed the LPS effects on MyD88, serum TNF-α and IL-6 concentration (p<0.001) which were reversed significantly by adding compound C. Conclusion: The results showed that heart is capable of producing TNF-α through TLR-4 and MyD88 activation independent of the classic immune system. Furthermore, AMPK activation followed by suppression of TLR activity can be considered as a target in protecting the failing heart in sepsis and MI and indicates a link between AMPK and TLR-4 in the heart.