Toll-like receptor 4 (TLR4) belongs to the family of microbial associated molecular patterns. TLR4 is involved in the immune response, and expressed in cardiomyocytes. Extensive activation of the immune response in the heart causes the release of proinflammatory cytokine mediators which depress heart function. High-mobility group box 1 (HMGB1), released under hypoxic conditions from both inflammatory cells and from cardiomyocytes, activates TLR4 signaling and may induce the release of proinflammatory cytokines and chemokines. The purpose of this research was: 1. To characterize the interaction between TLR4 and HMGB1 release to the medium. 2. To investigate whether the newly synthesized decoy peptide that resembles HMGB1 (HMP) binding domain is capable of reducing cardiomyocyte damage after hypoxia . Rat cardiomyocyte cell cultures were subjected to hypoxia with and without HMP (20 M). Lactate dehydrogenase (LDH), creatine kinase (CK), HMGB1 and TNFα secretion to the medium increased following hypoxia. Cardiomyocyte TLR4 and IκB markedly augmented while HMGB1 decreased in the cytosol (p<0.05). Silencing TLR4 with short interfering RNA against TLR4 dramatically reduced the hypoxic damage as revealed by LDH, CK and TNFα release. While 5M HMP had no effect on hypoxic cardiomyocytes, 20 M was efficient in reducing the damage markers. The decoy peptide protected the cardiomyocytes as seen by reduced CK and LDH levels released to the medium. TLR4 related damage is markedly reduced upon its silencing. These findings support the novel view that ischemia-induced HMGB1 release may be an important factor in the modulation of cardiac ischemic injury through TLR4 signaling.
