Study On The Upregulated Expression Of TLR2and TLR4by Extracellular HSP60in Cardiac Myocytes

Toll-like receptors (TLRs) play a critical role in immunological recognition of thepathogens, which exist in myocardial cells, vascular endothelial cells and neurons. There areat least11TLRs that have been cloned in humans, and in mouse, at least six TLRs namedTLR2/3/4/5/7/9are expressed in heart. Heat shock protein60(HSP60) is a member of HSPsfamily and about70-80%of them are located in mitochondria and cytoplasm in normal state.It has been reported that HSP60can be detected in the serum of rat models after LAD ligation（Lin et al, AJP,2007;293: H2238-47）.Our previous study demonstrated that the mRNAlevels of TLR2/4in rat H9c2cell-line and in ischemic myocardium were respectivelyincreased after exogenous HSP60treatment and ischemia induced by LAD ligation. Thus, wesupposed that the serum HSP60(extracellular HSP60) induced by myocardial ischemia notonly served as ligands of TLR2and TLR4, but also up-regulated the expression of TLR2andTLR4. In order to varify the hypothesis, we explored the mechanism of TLR2and TLR4up-regulation induced by exogenous HSP60in H9c2cells (in vitro) and the mechanism ofTLR2and TLR4up-regulation induced by endogenous HSP60in myocardial ischemia ratmodel (in vivo) and H9c2cells (in vitro).MethodIn cell culture supernatant of H9c2myocardial cells after exogenous HSP60treatment ormimic ischemia (using serum free, low glucose and low concentration of oxygen with1%O2medium), ELISA analysis was used to detect the concentration of HSP60. The expression of TLR2and TLR4receptor was detected by Western blot and Real-time PCR before and afterthe blockage of the major signal pathway molecule (TLR4, p38, JNK and NF-κB) of HSP60.Real-time PCR and Western blot were used to determine the production of TLR2andTLR4in rat model of myocardial induced by LAD ligation. The content of HSP60in serumwas determined by ELISA and western blot. Additionally, HSP60antibody was injected invein to neutralize serum HSP60, the expression of TLR2/4and the content of HSP60werealso detected.Result1. Exogenous HSP60up-regulated the expression of TLR2/4in normal cultured rat H9c2myocardium cell-line by TLR4-MyD88-JNK/NFκB signal pathway. The efficacy of increasedexpression of TLR2/4induced by exogenous HSP60was abolished after using signalinhibitors (TLR4siRNA, Inhi MyD88, SP600125, JNK inhibitor and NF-κB inhibitor(PDTC), which indicate that exogenous extracellular HSP60up-regulated the expression ofTLR2and TLR4by TLR4-MyD88-JNK/NFκB signal pathway. TLR4siRNA not onlyattenuated the expression of TLR4but also decrease the expression of TLR2and TLR4.However, TLR2siRNA only attenuated the expression of TLR2.2. HSP60was translocated to extracellular space after mimic ischemia treatment in H9c2cells. And the expression of TLR2/4was increased via TLR4-MyD88-JNK/NFκB signalpathway.3. HSP60was also detected in the serum of rat after myocardial ischemia modelinduceded by LAD ligation. Extracellular HSP60contributed to the increase of TLR2andTLR4. After4hours of LAD ligation, the content HSP60in serum was significant increased,but no change have been observed in sham operation group. Results from Western-blotindicated that both TLR2and TLR4up-regulated in several area in heart such as left ventricleanterior wall (LV-A), left ventricle posterior wall (LV-P), left ventricle and inter ventricular septum. After the injection of HSP60antibody in vein, myocardial ischemia model no longerup-regulated the expression of TLR2and TLR4, however, the injection of IgG antibody didnot have such effect.ConclusionsOur present study indicate that acute myocardial ischemia promote extracellular HSP60expression, which subsequently play a critical role in up-regulation of TLR2and TLR4byTLR4-MyD88-JNK/NFκB signal pathway.