Lipopolisaccharide-Induced Cardiac Inflammation And Left Ventricular Dysfunction: Role Of Toll-Like Receptor-4 In The Adult Heart

Objective LPS-induced cardiac dysfunction is an important risk factor of poor outcome of septic shock. The mechanism by which LPS induced myocardial dysfunction remains elusive. Systemic or compartmentalized over-production of inflammatory cytokines such as TNF-α and IL-1β may play important roles in the pathogenesis of LPS-induced myocardial depression. TLR4 is a newly found transmembrane receptor of LPS, which is responsible for LPS-induced inflammatory cytokines production of inflammatory cytokines. The purpose of this study was to demonstrate the importance of TLR4 in LPS-induced inflammatory cytokines expression in myocardium as well as in LPS-induced left ventricular depression in vivo.Materials and Methods1. Female TLR4-deficient mice C3H/HeJ and TLR4 wild-type mice C3H/HeN were injected intraperitoneally with (5mg/kg, n=5) Escherichia coli LPS. Control mice were injected with 200$l pyrogen-free saline. Clinical manifestation of mice following LPS administration were kept recorded at regular intervals for 24 hours while mortality of mice after LPS administration (25mg/kg, n=5) monitored for 36 hours. TNF-α and IL-1β were examined 2 hours after LPS treatment (n=5) while plasma concentrations of BUN, Cr, AST and blood sugar were determined 6 hours after LPS exposure (n=5) and arterial blood gas analyzed 12 hours later (n=5). TLR4 fragment containing mutation was amplified from the myocardium of C3H/HeJ mice for further sequence analysis.2. The expression of TNF-α and IL-1β in the hearts of C3H/HeJ and C3H/HeN mice were determined by RT-PCR and immunohistochemistry before and after LPS treatment. Hematoxylin and eosin staining and Masson staining were applied for routine histological examination. The changes of TLR4 mRNA and protein expression in the hearts of both mice before and after LPS treatment was also illustrated.3. LV function of mice was assessed by left ventricular puncture through diaphragm. Hematoxylin and eosin staining was applied for routine histological examination before LPS administration. Heart rate, LV pressure, +dp/dtmax and -dp/dtmax of C3H/HeN mice and C3H/HeJ were monitored before and 6 hours after LPS or saline injection.4. Statistical analysis were carried out by using SPSS 10 soft pack. Data were expressed as means ?SE. An unpaired t-test was used to assess differences in serum biochemical variables before and after LPS treatment and hemodynamic data between C3H/HeJ and C3H/HeN mice. ANOVA was used to determine significant differences in LPS-induced cytokine gene and protein expression at each time point. Statistical significance was set at p<0.05.Results1. C3H/HeN mice developed shock-like symptoms as early as 0.5 hours after LPS challenge, including ruffled hair, diarrhea, eye exudates, lethargy and loss of body weight. 50% of C3H/HeN mice died at 36 hours. A single i.p. injection of 5mg/kg LPS induced an early increase in TNF-α and IL-1β in serum of C3H/HeN mice at 2 hours following LPS administration. The increase of BUN, Cr, AST and blood sugar were observed at 6 hours after injection and a decrease of PaO2, SaO2, HCO3- and PH at 24 hours in C3H/HeN mice. In contrast, C3H/HeJ mice developed no shock-like syndrome and no C3H/HeJ mice died during the observation. There was no increase in the concentration of BUN, Cr, AST, blood sugar, TNF-α and IL-1β. Value of arterial blood gas remained normal. Sequence analysis of DNA fragment ofTLR4 amplified confirmed the point mutation of c to a in C3H/HeJ mice.2. TLR4 gene and protein expression was demonstrated by RT-PCR and immunohistochemistry in normal hearts of C3H/HeN and C3H/HeJ mice. LPS induced a robust and time-dependent increase in TNF-α and IL-1β mRNA transcripts in C3H/HeN mice with their maximum expression appearing at 0.5 hours and 4-6 hours respectively after LPS treatment. Immunohistochemistry study revealed a similar kinetics of TNF-α and IL-1β protein production with that of mRNA transcripts while their maximum being at 2 hours and 6 hours after LPS treatment r...