Protection against Myocardial Ischemia/Reperfusion Injury in TLR4-Deficient Mice Is Mediated through a Phosphoinositide 3-Kinase-Dependent Mechanism

Abstract

TLRs play a critical role in the induction of innate and adaptive immunity. However, TLRs have also been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We have reported that TLR4−/− mice show decreased myocardial injury following I/R; however, the protective mechanisms have not been elucidated. We examined the role of the PI3K/Akt signaling pathway in TLR4−/− cardioprotection following I/R injury. TLR4−/− and age-matched wild-type (WT) mice were subjected to myocardial ischemia for 45 min, followed by reperfusion for 4 h. Pharmacologic inhibitors of PI3K (wortmannin or LY294002) were administered 1 h before myocardial I/R. Myocardial infarct size/area at risk was reduced by 51.2% in TLR4−/− vs WT mice. Cardiac myocyte apoptosis was also increased in WT vs TLR4−/− mice following I/R. Pharmacologic blockade of PI3K abrogated myocardial protection in TLR4−/− mice following I/R. Specifically, heart infarct size/area at risk was increased by 98% in wortmannin and 101% in LY294002-treated TLR4−/− mice, when compared with control TLR4−/− mice. These data indicate that protection against myocardial I/R injury in TLR4−/− mice is mediated through a PI3K/Akt-dependent mechanism. The mechanisms by which PI3K/Akt are increased in the TLR4−/− myocardium may involve increased phosphorylation/inactivation of myocardial phosphatase and tensin homolog deleted on chromosome 10 as well as increased phosphorylation/inactivation of myocardial glycogen synthase kinase-3β. These data implicate innate immune signaling pathways in the pathology of acute myocardial I/R injury. These data also suggest that modulation of TLR4/PI3K/Akt-dependent signaling pathways may be a viable strategy for reducing myocardial I/R injury.

Activation of Toll-like receptor 4 signaling contributes to hippocampal neuronal death following global cerebral ischemia/reperfusion

“…Recent studies have shown that activation of the TLR4-mediated NFκB pathway plays a role in ischemia/reperfusion (I/R) injury. For example, we (Hua et al, 2007;Li et al, 2005) and others have reported that TLR4-mediated NFκB signaling contributes to myocardial ischemia/reperfusion injury and TLR4 deficiency protects the myocardium from ischemic injury. TLR4 is also involved in the pathogenesis of I/R injury in liver (Zhai et al, 2004), kidney (Kim et al, 2005) and lung tissues (Shimamoto et al, 2006), TLRs have been identified in the central nervous system (CNS) and are thought to play an important role in the brain's response to pathogens as well as toxic cell debris (Bottcher et al, 2003;Bsibsi et al, 2002;Maslinska et al, 2004) and inflammatory or autoimmune CNS diseases (Chakravarty and Herkenham, 2005;Kerfoot et al, 2004).…”

“…Nuclear proteins were isolated from hippocampal samples as previously described (Hua et al, 2005;Hua et al, 2007). NFκB binding activity was examined by EMSA in a 15 μl binding reaction mixture containing 15 μg of nuclear proteins and 35 fmol [γ-32 P] labeled doublestranded NFκB consensus oligonucleotide.…”

“…Cytoplasmic proteins were prepared from hippocampal tissues and immunoblots were performed as described previously (Hua et al, 2005;Hua et al, 2007). Briefly, the cellular proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto Hybond ECL membranes (Amersham Pharmacia, Piscataway, NJ).…”

Preconditioning with a TLR2 specific ligand increases resistance to cerebral ischemia/reperfusion injury

“…Cellular proteins were prepared from ischemic cerebral hemispheres (Hua et al 2007b), electrophoresed with SDS-polyacrylamide gel and transferred onto Hybond ECL membranes (Amersham Pharmacia, Piscataway, NJ) (Hua et al 2007a;Hua et al 2005;Hua et al 2007b). The ECL membranes were incubated with the appropriate primary antibody followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Inc.).…”

Inflammatory and fibrotic responses of cardiac fibroblasts to myocardial damage associated molecular patterns (DAMPs)

“…Knockout mouse studies have proven useful for identifying specific roles for DAMP receptors in modulating cardiac remodelling after injury or stress (summarised in Table 1). Global knockout or deficiency of TLR2 [34][35][36][37][38][39], TLR3 [40,41] and TLR4 [42][43][44][45][46][47][48][49][50][51][52][53][54][55][56] generally confers improvement of cardiac function and less adverse remodelling after injury, although detrimental effects have also been reported in TLR2 knockout mice [57]. In contrast to the perceived detrimental roles of TLRs 2, 3 and 4 in post-MI remodelling, a recent study suggested that TLR5 may play a beneficial role in protecting the heart from ischaemia/reperfusion injury [58].…”