Regulation and actions of activin A and follistatin in myocardial ischaemia-reperfusion injury.

Activin A, a member of the transforming growth factor-ß superfamily, is stimulated early in inflammation via the Toll-like receptor (TLR) 4 signalling pathway, which is also activated in myocardial ischaemia-reperfusion. Neutralising activin A by treatment with the activin-binding protein, follistatin, reduces inflammation and mortality in several disease models. This study assesses the regulation of activin A and follistatin in a murine myocardial ischaemia-reperfusion model and determines whether exogenous follistatin treatment is protective against injury. Myocardial activin A and follistatin protein levels were elevated following 30 min of ischaemia and 2h of reperfusion in wild-type mice. Activin A, but not follistatin, gene expression was also up-regulated. Serum activin A did not change significantly, but serum follistatin decreased. These responses to ischaemia-reperfusion were absent in TLR4(-/-) mice. Pre-treatment with follistatin significantly reduced ischaemia-reperfusion induced myocardial infarction. In mouse neonatal cardiomyocyte cultures, activin A exacerbated, while follistatin reduced, cellular injury after 3h of hypoxia and 2h of re-oxygenation. Neither activin A nor follistatin affected hypoxia-reoxygenation induced reactive oxygen species production by these cells. However, activin A reduced cardiomyocyte mitochondrial membrane potential, and follistatin treatment ameliorated the effect of hypoxia-reoxygenation on cardiomyocyte mitochondrial membrane potential. Taken together, these data indicate that myocardial ischaemia-reperfusion, through activation of TLR4 signalling, stimulates local production of activin A, which damages cardiomyocytes independently of increased reactive oxygen species. Blocking activin action by exogenous follistatin reduces this damage.

Inhibition of Toll-like receptor 4 with eritoran attenuates myocardial ischemia-reperfusion injury.

BACKGROUND: We previously reported that the functional mutation of Toll-like receptor 4 (TLR4) in C3H/HeJ mice subjected to myocardial ischemia-reperfusion (MI/R) injury resulted in an attenuation of myocardial infarction size. To investigate the ligand-activating TLR4 during MI/R injury, we evaluated the effect of eritoran, a specific TLR4 antagonist, on MI/R injury, with the goal of defining better therapeutic options for MI/R injury. METHODS AND RESULTS: C57BL/6 mice received eritoran (5 mg/kg) intravenously 10 minutes before 30 minutes of in situ of transient occlusion of the left anterior descending artery, followed by 120 minutes of reperfusion. Infarct size was measured using triphenyltetrazoliumchloride staining. A c-Jun NH(2)-terminal kinase (JNK) activation was determined by Western blotting, nuclear factor (NF)-kappaB activity was detected by gel-shift assay, and cytokine expression was measured by ribonuclease protection assay. Mice treated with eritoran developed significantly smaller infarcts when compared with mice treated with vehicle alone (21.0+/-6.4% versus 30.9+/-13.9%; P=0.041). Eritoran pretreatment resulted in a reduction in JNK phosphorylation (eritoran versus vehicle: 3.98+/-0.81 versus 7.01+/-2.21-fold increase; P=0.020), less nuclear NF-kappaB translocation (2.70+/-0.35 versus 7.75+/-0.60-fold increase; P=0.00007), and a decrease in cytokine expression (P<0.05). CONCLUSIONS: We conclude that inhibition of TLR4 with eritoran in an in situ murine model significantly reduces MI/R injury and markers of an inflammatory response.

Toll-like receptor 4 mediates ischemia/reperfusion injury of the heart.

BACKGROUND: Restoration of blood flow to the ischemic heart may paradoxically exacerbate tissue injury (ischemia/reperfusion injury). Toll-like receptor 4, expressed on several cell types, including cardiomyocytes, is a mediator of the host inflammatory response to infection. Because ischemia/reperfusion injury is characterized by an acute inflammatory reaction, we investigated toll-like receptor 4 activation in a murine model of regional myocardial ischemia/reperfusion injury. We used C3H/HeJ mice, which express a nonfunctional toll-like receptor 4, to assess the pertinence of this receptor to tissue injury after reperfusion of ischemic myocardium. METHODS: Wild-type mice (C3H/HeN) or toll-like receptor 4 mutant mice (C3H/HeJ) were subjected to 60 minutes of regional myocardial ischemia followed by 2 hours of reperfusion. At the end of reperfusion, the area at risk and the myocardial infarct size were measured as the end point of myocardial ischemia/reperfusion injury. Myocardial mitogen-activated protein kinase activation was measured by Western blotting, and nuclear translocation of nuclear factor-kappaB and activator protein-1 was determined by electrophoretic mobility shift assay. Ischemia/reperfusion-injured myocardium was also assessed by ribonuclease protection assay for expression of inflammatory mediators (tumor necrosis factor-alpha, interleukin-1beta, monocyte chemotactic factor-1, and interleukin-6). RESULTS: The area at risk was similar for all groups after myocardial ischemia/reperfusion injury. There was a 40% reduction in infarct size (as a percentage of the area at risk) in C3H/HeJ mice compared with C3H/HeN mice (P =.001). Within the myocardium, significant activation of c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase was observed in both strains after ischemia and during reperfusion as compared with an absence of mitogen-activated protein kinase activation during sham operations; however, c-Jun N-terminal kinase activity, but not p38 or extracellular signal-regulated kinase activity, was significantly reduced in C3H/HeJ mice (P <.05). In both groups, nuclear factor-kappaB and activator protein-1 nuclear translocation occurred in the myocardium during myocardial ischemia/reperfusion injury, but, by densitometric analysis, nuclear translocation of nuclear factor-kappaB and activator protein-1 was significantly decreased in C3H/HeJ mice compared with C3H/HeN mice. Interleukin-1beta, monocyte chemotactic factor-1, and interleukin-6 were detectable in reperfused ischemic myocardium but were not detected in sham-operated myocardium; the expression of each of these mediators was significantly decreased in the myocardial tissue of C3H/HeJ mice when compared with expression in the control C3H/HeN mouse strain. CONCLUSIONS: Our data suggest that toll-like receptor 4 may mediate, at least in part, myocardial ischemia/reperfusion injury. Inhibition of toll-like receptor 4 activation may be a potential therapeutic target to attenuate ischemia/reperfusion-induced tissue damage in the clinical setting.

FR167653 diminishes infarct size in a murine model of myocardial ischemia-reperfusion injury.

OBJECTIVE: During myocardial ischemia-reperfusion injury, p38 mitogen-activated protein kinase is activated. We examined the effect of a highly specific inhibitor of p38 mitogen-activated protein kinase, FR167653, in an experimental model of regional myocardial ischemia-reperfusion. METHODS: CD-1 mice received FR167653 intraperitoneally 24 hours before 30 minutes of transient occlusion of the left anterior descending artery, followed by 120 minutes of reperfusion. The p38 mitogen-activated protein kinase activation and kinase activity were determined by Western blotting with monoclonal antibodies for the phosphorylated from of p38 mitogen-activated protein kinase or its substrate, activating transcription factor 2. Nuclear factor kappaB activity was measured by detecting translocation of nuclear factor kappaB to the nucleus. The expression of inflammatory cytokines was measured by ribonuclease protection assay. RESULTS: Pretreatment of mice with FR167653 before myocardial ischemia-reperfusion resulted in a reduction in p38 mitogen-activated protein kinase phosphorylation (P =.018), an inhibition of p38 mitogen-activated protein kinase activity (P =.047), a smaller amount of nuclear factor kappaB (P =.001), and a decrease in the expression of inflammatory cytokines (tumor necrosis factor alpha: P =.023, interleukin 1beta: P =.038, monocyte chemotactic protein 1: P =.0001) in the heart and the development of a significantly smaller infarct (P =.0069) relative to hearts from mice treated with vehicle alone. Activation of c-Jun N-terminal kinase and extracellular signal-regulated kinase were observed after myocardial ischemia-reperfusion without inhibition by FR167653. CONCLUSION: We conclude that FR167653 selectively inhibits p38 mitogen-activated protein kinase activation and activity during regional myocardial ischemia-reperfusion injury and efficaciously reduces infarct size (by 73.6%). Thus p38 mitogen-activated protein kinase inhibition may have a role in the treatment of myocardial ischemia-reperfusion.

HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse hearts.

We investigated the role of inducible heat shock proteins 70.1 and 70.3 (HSP70.1 and HSP70.3, respectively) in myocardial ischemic preconditioning (IP) in mice. Wild-type (WT) mice and HSP70.1- and HSP70.3-null [HSP70.1/3(-/-)] mice were subjected to IP and examined 24 h later during the late phase of protection. IP significantly increased steady-state levels of HSP70.1 and HSP70.3 mRNA and expression of inducible HSP70 protein in WT myocardium. To assess protection against tissue injury, mice were subjected to 30 min of regional ischemia and 3 h of reperfusion. In WT mice, IP reduced infarct size by 43% compared with sham IP-treated mice. In contrast, IP did not reduce infarct size in HSP70.1/3(-/-) mice. Absence of inducible HSP70.1 and HSP70.3 had no effect, however, on classical or early-phase protection produced by IP, which significantly reduced infarct size in HSP70.1/3(-/-) mice. We conclude that inducible HSP70.1 and HSP70.3 are required for late-phase protection against infarction following IP in mice.