Pascal Knuefermann

In Vivo Expression of Proinflammatory Mediators in the Adult Heart after Endotoxin Administration: The Role of Toll-Like Receptor–4

Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and nitric oxide (NO) may play a role in lipopolysaccharide (LPS)-induced cardiac depression. Toll-like receptor-4 (TLR-4) mediates the cytokine response to LPS in immune cells. TLR-4 also is expressed in human and murine myocardial tissue. Therefore, the hypothesis that LPS induces proinflammatory cytokines in the heart via TLR-4 was tested. C3H/HeJ (TLR-4 deficient) and C3HeB/FeJ mice were studied. LPS induced a robust increase in myocardial TNF-alpha and IL-1beta mRNA in C3HeB/FeJ mice. The response in C3H/HeJ mice was blunted and delayed. Myocardial TNF-alpha and IL-1beta protein levels were higher in C3HeB/FeJ mice, as were inducible NO synthase protein and NO production. Activation of myocardial NF-kappaB was observed within 30 min in C3HeB/FeJ mice but not in C3H/HeJ mice. These findings suggest that myocardial TLR-4 is involved in signaling cytokine production within the heart during endotoxic shock.

Nuclear Factor-κB Protects the Adult Cardiac Myocyte Against Ischemia-Induced Apoptosis in a Murine Model of Acute Myocardial Infarction

Background—Previous studies have shown that tumor necrosis factor (TNF) confers cytoprotective responses in cardiac myocytes. However, the mechanisms for the cytoprotective effects of TNF remain unknown. Given that TNF signals through nuclear factor &kgr;B (NF-&kgr;B) and given that NF-&kgr;B mediates cytoprotective responses, we asked whether NF-&kgr;B activation conferred cytoprotective responses in acute myocardial ischemia/infarction. Methods and Results—We examined infarct size and the prevalence of apoptosis in transgenic mice harboring cardiac-restricted expression of a mutated I&kgr;B&agr; protein (I&kgr;B&agr;&Dgr;N) that prevents nuclear translocation of NF-&kgr;B in cardiac myocytes. Triphenyltetrazolium chloride staining showed that infarct size was ≈50% greater (P <0.02) in the I&kgr;B&agr;&Dgr;N mice compared with littermate controls at 24 hours. The prevalence of cardiac myocyte apoptosis was significantly greater (P <0.008) in the I&kgr;B&agr;&Dgr;N mice compared with the littermate control mice 3 and 6 hours after left anterior descending occlusion. To explore the mechanism for these findings, we examined protein levels of c-IAP1, c-IAP2, and Bcl-2 as well as manganese superoxide dismutase and c-Jun NH2-terminal kinase activity. These studies showed that protein levels of c-IAP1 and Bcl-2 were significantly lower in the I&kgr;B&agr;&Dgr;N mice, whereas there was no change in c-IAP2 levels, manganese superoxide dismutase, or c-Jun NH2-terminal kinase activity. Conclusions—Transgenic mice with a defect in activation of NF-&kgr;B have increased susceptibility to tissue injury after acute left anterior descending occlusion. These studies suggest that the cytoprotective effects of NF-&kgr;B are mediated, at least in part, by Bcl-2 or c-IAP1.

Load-Dependent and -Independent Regulation of Proinflammatory Cytokine and Cytokine Receptor Gene Expression in the Adult Mammalian Heart

Background—Although previous studies have examined the effects of acute hemodynamic pressure overload on proinflammatory cytokine gene expression, the effects of sustained hemodynamic overloading have not been examined. Methods and Results—Sustained hemodynamic pressure overloading was produced in mice by transverse constriction of the aorta. Proinflammatory cytokine and cytokine receptor gene expression were determined by ribonuclease protection assays (RPA) at 6 hours and at 3, 7, 14 and 35 days after banding. M-mode echocardiography was used to assess left ventricular structure and function at identical time points. RPA showed that tumor necrosis factor (TNF), interleukin (IL)-1&bgr;, and IL-6 mRNA levels were maximal at 6 hours and returned to baseline levels within 72 hours. There was a significant increase in IL-1RII and IL-6R&agr; receptor mRNA levels after overloading but no significant increase in TNFR1, TNFR2, IL-1RI, or gp130 mRNA levels. The transient increase in expression of proinflammatory cytokine gene expression was not explained by changes in left ventricular loading conditions, left ventricular wall stress, desensitization of proinflammatory genes, or decreased nuclear factor-&kgr;B activation. It is interesting that transverse constriction of the aorta provoked an increase in the expression of tristetraprolin, a homeostatic zinc finger protein that is known to destabilize TNF mRNA. Conclusion—Sustained hemodynamic overloading provokes a transient increase in proinflammatory cytokine and cytokine receptor gene expression; however, the decrease in proinflammatory cytokine gene expression occurred in the absence of changes in loading conditions, suggesting that the expression of proinflammatory cytokines in the heart is regulated, at least in part, by load-dependent and load-independent mechanisms.

CD14-Deficient Mice Are Protected Against Lipopolysaccharide-Induced Cardiac Inflammation and Left Ventricular Dysfunction

Background—The molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined. CD14 mediates the inflammatory response to lipopolysaccharide (LPS) in various organs including the heart. In this study we investigated the role of CD14 in LPS-induced myocardial dysfunction in vivo. Methods and Results—Wild-type and CD14-deficient (CD14-D) mice were challenged with Escherichia coli LPS. Myocardial tumor necrosis factor, interleukin-1&bgr; (IL-1&bgr;), and NOS2 induction was measured before and 6 hours after LPS challenge. Echocardiographic parameters of left ventricular function were measured before and 6 hours after LPS administration. LPS challenge induced a significant increase in myocardial tumor necrosis factor and IL-1&bgr; mRNA and protein expression in wild-type mice. In contrast, mRNA and protein levels for TNF and IL-1&bgr; were significantly blunted in CD14-D mice. An increase in NOS2 protein was noted within 6 hours of LPS provocation only in the hearts of wild-type mice. This was associated with an increase in ventricular cGMP levels. Activation of nuclear factor-&kgr;B was observed within 30 minutes of LPS in the hearts of wild-type mice but not in CD14-D mice. In wild-type mice, LPS significantly decreased left ventricular fractional shortening, velocity of circumferential shortening, and dP/dtmax. LPS-treated CD14-D mice maintained normal cardiac function. Conclusions—These results suggest that CD14 is important in mediating the proinflammatory response induced by LPS in the heart and that CD14 is necessary for the development of left ventricular dysfunction during LPS-induced shock in vivo.

Development of murine ischemic cardiomyopathy is associated with a transient inflammatory reaction and depends on reactive oxygen species

We examined the effects of daily repetitive brief (15 min) myocardial ischemia and reperfusion (I/R) in WT C57/BL6 and extracellular superoxide dismutase (EC-SOD)-overexpressing mice. In the absence of myocardial necrosis, I/R resulted in persistent fibrosis in ischemic areas of C57/BL6 mice associated with persistent global and segmental anterior wall dysfunction. The I/R protocol induced chemokines (peak 3 days) followed sequentially by infiltration of macrophages and myofibroblasts (5 days). Fibrosis peaked at 7 days and was stable at 28 days despite regression of the chemokine and cellular response. Discontinuation of I/R at 7 or 28 days led to regression of fibrosis and ventricular dysfunction. In contrast, the EC-SOD mice developed markedly less chemokine induction, cell response, and fibrosis, with no ventricular dysfunction. Reversible fibrosis and ventricular dysfunction are features of human hibernating myocardium. The reduction of the cellular and functional response in EC-SOD mice suggests a role for reactive O2 in the pathogenesis of ischemic cardiomyopathy.

TOLL-LIKE RECEPTOR 4, NITRIC OXIDE, AND MYOCARDIAL DEPRESSION IN ENDOTOXEMIA

The molecular mechanisms that mediate gram-negative sepsis-associated myocardial dysfunction remain elusive. Myocardial expression of inflammatory mediators is Toll-like receptor 4 (TLR4) dependent. However, it remains to be elucidated whether TLR4, expressed on cardiac myocytes, mediates impairment of cardiac contractility after lipopolysaccharide (LPS) application. Cardiac myocyte contractility, measured as sarcomere shortening of isolated cardiac myocytes from C3H/HeJ (with nonfunctional TLR4) and C3H/HeN (control), were recorded at stimulation frequencies between 0.5 and 10 Hz and after incubation with 1 and 10 μg/mL LPS for up to 8 h. Control cells treated with LPS were investigated with and without a competitive LPS inhibitor (E5564) and a specific inducible nitric oxide synthase (iNOS) inhibitor S-methylisothiourea. In control mice, LPS reduced sarcomere shortening amplitude and prolonged duration of relaxation, whereas sarcomere shortening of C3H/HeJ cells was insensitive to LPS. NFκB and iNOS were upregulated after LPS application in control mice compared with C3H/HeJ. Inhibition of TLR4 by E5564 as well as inhibition of iNOS prevented the influence of LPS on contractile activity in control myocytes. LPS-dependent suppression of cardiac myocyte contractility was significantly blunted in C3H/HeJ mice. Competitive inhibition of functional TLR4 with E5564 protects cardiac myocyte contractility against LPS. These findings suggest that TLR4, expressed on cardiac myocytes, contributes to sepsis-induced myocardial dysfunction. E5564, currently under investigation in two clinical phase II trials, seems to be a new therapeutic option for the treatment of myocardial dysfunction in sepsis associated with endotoxemia.

Toll-Like Receptor 2 Mediates Staphylococcus aureus–Induced Myocardial Dysfunction and Cytokine Production in the Heart

Background—Staphylococcus aureus sepsis is associated with significant myocardial dysfunction. Toll-like receptor 2 (TLR2) mediates the inflammatory response to S aureus and may trigger an innate immune response in the heart. We hypothesized that a TLR2 deficiency would attenuate S aureus–induced cardiac proinflammatory mediator production and the development of cardiac dysfunction. Methods and Results—Wild-type and TLR2-deficient (TLR2D) mice were studied. S aureus challenge significantly increased tumor necrosis factor, interleukin-1&bgr;, and nitric oxide expression in hearts of wild-type mice. This response was significantly blunted in TLR2D mice. Hearts from TLR2D mice had impaired S aureus–induced activation of interleukin-1 receptor–associated kinase, c-Jun NH2 terminal kinase, nuclear factor-&kgr;B, and activator protein-1. Moreover, hearts from TLR2D mice were protected against S aureus–induced contractile dysfunction. Conclusions—These results show for the first time that TLR2 signaling contributes to the loss of myocardial contractility and cytokine production in the heart during S aureus sepsis.

Cytokines as emerging targets in the treatment of heart failure.

Recent studies have identified the importance of biologically active molecules such as neurohormones in disease progression in heart failure. More recently it has become apparent that in addition to neurohormones another portfolio of biologically active molecules termed cytokines are also expressed in the setting of heart failure. This article reviews recent clinical and experimental material which suggest that the cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) may represent another class of biologically active molecules that are responsible for the development and progression of heart failure. In addition, we also review the early results from clinical trials that have utilized various targeted anti-cytokine strategies in patients with heart failure.

Toll-like receptor 4 deficiency: Smaller infarcts, but nogain in function

It has been reported that Toll-like receptor 4 (TLR4) deficiency reduces infarct size after myocardial ischemia/reperfusion (MI/R). However, measurement of MI/R injury was limited and did not include cardiac function. In a chronic closed-chest model we assessed whether cardiac function is preserved in TLR4-deficient mice (C3H/HeJ) following MI/R, and whether myocardial and systemic cytokine expression differed compared to wild type (WT). Infarct size (IS) in C3H/HeJ assessed by TTC staining after 60 min ischemia and 24h reperfusion was significantly smaller than in WT. Despite a smaller infarct size, echocardiography showed no functional difference between C3H/HeJ and WT. Left-ventricular developed pressure measured with a left-ventricular catheter was lower in C3H/HeJ (63.0 ± 4.2 mmHg vs. 77.9 ± 1.7 mmHg in WT, p < 0.05). Serum cytokine levels and myocardial IL-6 were higher in WT than in C3H/HeJ (p < 0.05). C3H/HeJ MI/R showed increased myocardial IL-1β and IL-6 expression compared to their respective shams (p < 0.05), indicating TLR4-independent cytokine activation due to MI/R. These results demonstrate that, although a mutant TLR4 signaling cascade reduces myocardial IS and serum cytokine levels, it does not preserve myocardial function. The change in inflammatory response, secondary to a non-functional TLR-4 receptor, may contribute to the observed dichotomy between infarct size and function in the TLR-4 mutant mouse.BackgoundIt has been reported that Toll-like receptor 4 (TLR4) deficiency reduces infarct size after myocardial ischemia/reperfusion (MI/R). However, measurement of MI/R injury was limited and did not include cardiac function. In a chronic closed-chest model we assessed whether cardiac function is preserved in TLR4-deficient mice (C3H/HeJ) following MI/R, and whether myocardial and systemic cytokine expression differed compared to wild type (WT).ResultsInfarct size (IS) in C3H/HeJ assessed by TTC staining after 60 min ischemia and 24h reperfusion was significantly smaller than in WT. Despite a smaller infarct size, echocardiography showed no functional difference between C3H/HeJ and WT. Left-ventricular developed pressure measured with a left-ventricular catheter was lower in C3H/HeJ (63.0 ± 4.2 mmHg vs. 77.9 ± 1.7 mmHg in WT, p < 0.05). Serum cytokine levels and myocardial IL-6 were higher in WT than in C3H/HeJ (p < 0.05). C3H/HeJ MI/R showed increased myocardial IL-1β and IL-6 expression compared to their respective shams (p < 0.05), indicating TLR4-independent cytokine activation due to MI/R.ConclusionThese results demonstrate that, although a mutant TLR4 signaling cascade reduces myocardial IS and serum cytokine levels, it does not preserve myocardial function. The change in inflammatory response, secondary to a non-functional TLR-4 receptor, may contribute to the observed dichotomy between infarct size and function in the TLR-4 mutant mouse.

Bacterial DNA induces myocardial inflammation and reduces cardiomyocyte contractility: role of Toll-like receptor 9

AIMS Myocardial function is severely compromised during sepsis. Several underlying mechanisms have been proposed. The innate immune system, i.e. toll-like receptor (TLR) 2 and 4, significantly contributes to cardiac dysfunction. Little is known regarding TLR9 and its pathogenic ligand bacterial DNA in the myocardium. We therefore studied the role of TLR9 in myocardial inflammation and cardiac contractility. METHODS AND RESULTS Wild-type (WT, C57BL/6) and TLR9-deficient (TLR9-D) mice and isolated cardiomyocytes were challenged with synthetic bacterial DNA (CpG-ODN). Myocardial contractility as well as markers of inflammation/signalling were determined. Isolated cardiomyocytes incorporated fluorescence-marked CpG-ODN. In WT mice, CpG-ODN caused a robust response in hearts demonstrated by increased levels of tumour necrosis factor (TNF-alpha), interleukin (IL)-1beta, IL-6, inducible nitric oxide synthase (iNOS), and nuclear factor kappaB activity. This inflammatory response was absent in TLR9-D mice. Under similar conditions, contractility measurements of isolated ventricular cardiomyocytes demonstrated a TLR9-dependent loss of sarcomeric shortening after CpG-ODN exposure. This observation was iNOS dependent as the application of a specific iNOS inhibitor reversed sarcomeric shortening to normal levels. CONCLUSION Our data suggest that bacterial DNA contributes to myocardial cytokine production and loss of cardiomyocyte contractility via TLR9.