Synthetic CpG oligonucleotides induce a genetic profile ameliorating murine myocardial I/R injury.

We previously demonstrated that pre-conditioning with CpG oligonucleotide (ODN) 1668 induces quick up-regulation of gene expression 3 hours post-murine myocardial ischaemia/reperfusion (I/R) injury, terminating inflammatory processes that sustain I/R injury. Now, performing comprehensive microarray and biocomputational analyses, we sought to further enlighten the "black box" beyond these first 3 hours. C57BL/6 mice were pretreated with either CpG 1668 or with control ODN 1612, respectively. Sixteen hours later, myocardial ischaemia was induced for 1 hour in a closed-chest model, followed by reperfusion for 24 hours. RNA was extracted from hearts, and labelled cDNA was hybridized to gene microarrays. Data analysis was performed with BRB ArrayTools and Ingenuity Pathway Analysis. Functional groups mediating restoration of cellular integrity were among the top up-regulated categories. Genes known to influence cardiomyocyte survival were strongly induced 24 hours post-I/R. In contrast, proinflammatory pathways were down-regulated. Interleukin-10, an upstream regulator, suppressed specifically selected proinflammatory target genes at 24 hours compared to 3 hours post-I/R. The IL1 complex is supposed to be one regulator of a network increasing cardiovascular angiogenesis. The up-regulation of numerous protective pathways and the suppression of proinflammatory activity are supposed to be the genetic correlate of the cardioprotective effects of CpG 1668 pre-conditioning.

Tlr2 deficiency does not limit the development of left ventricular hypertrophy in a model of transverse aortic constriction induced pressure overload.

BACKGROUND: Toll-like receptors (TLRs) are involved in a variety of cardiovascular disorders, including septic cardiomyopathy, ischemia/reperfusion, heart failure, and cardiac hypertrophy. Previous research revealed that TLR4 promotes cardiac hypertrophy in vivo. Therefore, we investigated whether TLR2 is also involved in the development of cardiac hypertrophy. METHODS: Tlr2 deficient and wild type mice were subjected to transverse aortic constriction (TAC) or sham operation procedure. Left ventricular, heart and lung weights as well as hemodynamic parameters were determined after 3, 14 or 28 days. Real-time RT PCR was used to evaluate left ventricular gene expression. Protein content was determined via ELISA. RESULTS: TAC increased systolic left ventricular pressure, contraction and relaxations velocities as well as the heart weight in both genotypes. Tlr2 deficiency significantly enhanced cardiac hypertrophy after 14 and 28 days of TAC. Left ventricular end-diastolic pressure and heart rate increased in Tlr2(-/-) TAC mice only. Fourteen days of TAC led to a significant elevation of ANP, BNP, TGFß and TLR4 mRNA levels in Tlr2(-/-) left ventricular tissue. CONCLUSION: These data suggest that Tlr2 deficiency may promote the development of cardiac hypertrophy and ventricular remodeling after transverse aortic constriction.

Ultrasound confirmation of central venous catheter position via a right supraclavicular fossa view using a microconvex probe: an observational pilot study.

BACKGROUND: Visualisation of a central venous catheter (CVC) with ultrasound is restricted to the internal jugular vein (IJV). CVC tip position is confirmed by chest radiography, intracardiac ECG or transoesophageal/transthoracic echocardiography (TEE/TTE). OBJECTIVE: We explored the feasibility, safety and accuracy of a right supraclavicular view for visualisation of the lower superior vena cava (SVC) and the right pulmonary artery (RPA) as an ultrasound landmark for real-time ultrasound-guided CVC tip positioning via the right IJV. Ultrasound was then compared with chest radiography. DESIGN: An observational pilot study. SETTING: Bonn, University Hospital, Germany. From July to October 2012. PATIENTS: Fifty-one patients scheduled for elective surgery. Reasons for exclusion were emergency procedure, thrombosis or small IJV lumen and mechanical obstacle to guidewire advancement. INTERVENTION: In 48 patients, CVC insertion via the right IJV and progress of the guidewire into the lower SVC were continuously guided by an ultrasound transducer in the right supraclavicular fossa. MAIN OUTCOME MEASURES: CVC tip position in lower SVC and tip-to-carina distance were assessed with chest radiography as a reference method and additionally with TEE in cardiothoracic patients. Insertion depth was compared with intracardiac ECG and body-height formula. RESULTS: The guidewire tip was seen in the SVC of all patients. In four patients, the tip was not visible in proximity of the RPA. Chest radiography and TEE confirmed CVC tip position in the lower SVC (zone A). Bland-Altman analysis revealed an average of difference of 1.6 cm for ultrasound versus ECG (95% limit of agreement -2 to 5 cm) and an average of difference of 1 cm for ultrasound versus body-height formula (95% limit of agreement -2 to 4 cm). CONCLUSION: Ultrasound via a right supraclavicular view is a feasible, well tolerated and accurate approach and should be further explored. Chest radiography confirmed CVC position in the lower SVC.

Toll-like receptor 9 promotes cardiac inflammation and heart failure during polymicrobial sepsis.

BACKGROUND: Aim was to elucidate the role of toll-like receptor 9 (TLR9) in cardiac inflammation and septic heart failure in a murine model of polymicrobial sepsis. METHODS: Sepsis was induced via colon ascendens stent peritonitis (CASP) in C57BL/6 wild-type (WT) and TLR9-deficient (TLR9-D) mice. Bacterial load in the peritoneal cavity and cardiac expression of inflammatory mediators were determined at 6, 12, 18, 24, and 36 h. Eighteen hours after CASP cardiac function was monitored in vivo. Sarcomere length of isolated cardiomyocytes was measured at 0.5 to 10 Hz after incubation with heat-inactivated bacteria. RESULTS: CASP led to continuous release of bacteria into the peritoneal cavity, an increase of cytokines, and differential regulation of receptors of innate immunity in the heart. Eighteen hours after CASP WT mice developed septic heart failure characterised by reduction of end-systolic pressure, stroke volume, cardiac output, and parameters of contractility. This coincided with reduced cardiomyocyte sarcomere shortening. TLR9 deficiency resulted in significant reduction of cardiac inflammation and a sustained heart function. This was consistent with reduced mortality in TLR9-D compared to WT mice. CONCLUSIONS: In polymicrobial sepsis TLR9 signalling is pivotal to cardiac inflammation and septic heart failure.

Remote ischaemic preconditioning for heart surgery. The study design for a multi-center randomized double-blinded controlled clinical trial--the RIPHeart-Study.

AIMS: Transient ischaemia of non-vital tissue has been shown to enhance the tolerance of remote organs to cope with a subsequent prolonged ischaemic event in a number of clinical conditions, a phenomenon known as remote ischaemic preconditioning (RIPC). However, there remains uncertainty about the efficacy of RIPC in patients undergoing cardiac surgery. The purpose of this report is to describe the design and methods used in the "Remote Ischaemic Preconditioning for Heart Surgery (RIPHeart)-Study". METHODS: We are conducting a prospective, randomized, double-blind, multicentre, controlled trial including 2070 adult cardiac surgical patients. All types of surgery in which cardiopulmonary bypass is used will be included. Patients will be randomized either to the RIPC group receiving four 5 min cycles of transient upper limb ischaemia/reperfusion or to the control group receiving four cycles of blood pressure cuff inflation/deflation at a dummy arm. The primary endpoint is a composite outcome (all-cause mortality, non-fatal myocardial infarction, any new stroke, and/or acute renal failure) until hospital discharge. CONCLUSION: The RIPHeart-Study is a multicentre trial to determine whether RIPC may improve clinical outcome in cardiac surgical patients.

Sinus venous thrombosis: a differential diagnosis of postpartum headache.

In this report, we describe a patient who developed severe headache following epidural analgesia for labor and delivery. Although the epidural puncture had been reported to be uneventful, headache was initially suspected to result from an accidental dural puncture. After the headache worsened, a sinus venous thrombosis was suspected and subsequently confirmed by magnetic resonance imaging. This case highlights the difficulty of differential diagnosis of headache in the postnatal period in patients after EDA and stresses the necessity of considering alternative pathologies.

Low-tidal-volume prevent ventilation induced inflammation in a mouse model of sepsis.

BACKGROUND AND GOAL OF THE STUDY: Pulmonary inflammation, increased vascular permeability, and pulmonary edema, occur in response to primary pulmonary infections like pneumonia but are also evident in endotoxemia or sepsis. Mechanical ventilation augments pre-existing lung injury and inflammation resulting from exposure to microbial products. The objective of this study was to test the hypothesis that low-tidal-volume prevent ventilation induced lung injury in sepsis. MATERIALS AND METHODS: 10-12-week-old male C57BL/6N-mice received an intraperitoneal (i.p.) injection with equipotent dosages of LPS, 1668-thioate, 1612-thioate, or PBS. 120 min after injection, mice were randomized to low- (LV, 7 ± 1 ml/kg) or high-tidal-volume (HV, 25 ± 1 ml/kg) ventilation. Hemodynamic and ventilatory parameters were recorded and inflammatory markers were analyzed form BAL that was generated after 90 minute ventilation. RESULTS AND DISCUSSION: Arterial blood pressures declined during mechanical ventilation in all groups. pO2 decreased in LPS injected and CO2 increased in sham, LPS, and 1612-thioate administered mice at 45 min and in 1668-thioate injected mice after 90 minute LV ventilation compared to respective HV groups. BAL protein concentrations increased in HV ventilated and 1668- or 1612-thioat pre-treated mice. BAL TNF-α protein concentrations increased in both LPS- and 1668-thioate-injected and IL-1ß protein concentrations only in LPS-injected and HV ventilated mice. Most notably, no increased protein concentrations were observed in any of the LV ventilated groups. CONCLUSION: We conclude that low-tidal-volume ventilation may be a potential strategy for the prevention of ventilator induced lung injury in a murine model of systemic TLR agonist induced lung injury.

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.

Improve hip fracture outcome in the elderly patient (iHOPE): a study protocol for a pragmatic, multicentre randomised controlled trial to test the efficacy of spinal versus general anaesthesia.

INTRODUCTION: Hip fracture surgery is associated with high in-hospital and 30-day mortality rates and serious adverse patient outcomes. Evidence from randomised controlled trials regarding effectiveness of spinal versus general anaesthesia on patient-centred outcomes after hip fracture surgery is sparse. METHODS AND ANALYSIS: The iHOPE study is a pragmatic national, multicentre, randomised controlled, open-label clinical trial with a two-arm parallel group design. In total, 1032 patients with hip fracture (>65 years) will be randomised in an intended 1:1 allocation ratio to receive spinal anaesthesia (n=516) or general anaesthesia (n=516). Outcome assessment will occur in a blinded manner after hospital discharge and inhospital. The primary endpoint will be assessed by telephone interview and comprises the time to the first occurring event of the binary composite outcome of all-cause mortality or new-onset serious cardiac and pulmonary complications within 30 postoperative days. In-hospital secondary endpoints, assessed via in-person interviews and medical record review, include mortality, perioperative adverse events, delirium, satisfaction, walking independently, length of hospital stay and discharge destination. Telephone interviews will be performed for long-term endpoints (all-cause mortality, independence in walking, chronic pain, ability to return home cognitive function and overall health and disability) at postoperative day 30±3, 180±45 and 365±60. ETHICS AND DISSEMINATION: iHOPE has been approved by the leading Ethics Committee of the Medical Faculty of the RWTH Aachen University on 14 March 2018 (EK 022/18). Approval from all other involved local Ethical Committees was subsequently requested and obtained. Study started in April 2018 with a total recruitment period of 24 months. iHOPE will be disseminated via presentations at national and international scientific meetings or conferences and publication in peer-reviewed international scientific journals. TRIAL REGISTRATION NUMBER: DRKS00013644; Pre-results.

Toll-like receptor 9 expression in murine and human adrenal glands and possible implications during inflammation.

CONTEXT: Sepsis is a leading cause of death in the Western world and can be associated with failure of the hypothalamic-pituitary-adrenal axis. A coordinated response of the adrenal and immune system is of vital importance for survival during sepsis. Within the immune response, Toll-like receptors (TLRs) play a crucial role by recognizing pathogen-associated molecules such as bacterial DNA. TLR-9 can detect motifs of unmethylated cytosine-phosphate-guanine (CpG) dinucleotides (CpG-DNA) being present in bacterial DNA. OBJECTIVE: We investigated whether TLR-9 is expressed in human and murine adrenal glands and whether its activation is associated with an adrenal response. DESIGN: Human fetal and adult adrenal glands; wild-type, C57BL/6 and TLR-9 deficient (TLR-9-/-) mice; and in vitro cell line models were used in the study. SETTING: The study took place at a university hospital. RESULTS: TLR-9 is expressed in human and murine adrenal glands, as well as in in vitro cell lines (Y-1 and NCI-H295R cells). CpG-oligodeoxynucleotide challenge caused a 3-fold increase in plasma levels of corticosterone in wild-type mice. This effect was not observed in TLR-9-/- mice. Furthermore, CpG-oligodeoxynucleotide challenge resulted in a strong release of several inflammatory cytokines, such as TNF-alpha, and IL-1beta, -6, -10, and -12 in vivo as well as in vitro. Again, this effect was not present in TLR-9-/- mice. CONCLUSIONS: TLR-9 is present in both murine and human adrenal glands. TLR-9 stimulation led to a corticosterone and inflammatory cytokine response. TLR-9 may play a role in the regulation of the hypothalamic-pituitary-adrenal axis during conditions in which bacterial DNA is present.

CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo.

BACKGROUND: Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-alpha) and interleukin-1beta (IL-1beta). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo. METHODS: Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured. RESULTS: In WT mice, CpG-ODN induced a strong activation of pulmonary NFkappaB as well as a significant increase in pulmonary TNF-alpha and IL-1beta mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice. CONCLUSION: This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.

Toll-like receptor 4 deficiency: smaller infarcts, but no gain in function.

BACKGROUND: 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). RESULTS: 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-1beta and IL-6 expression compared to their respective shams (p < 0.05), indicating TLR4-independent cytokine activation due to MI/R. CONCLUSION: 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.

Decreased Secondary Lesion Growth and Attenuated Immune Response after Traumatic Brain Injury in Tlr2/4-/- Mice.

Danger-associated molecular patterns are released by damaged cells and trigger neuroinflammation through activation of non-specific pattern recognition receptors, e.g., toll-like receptors (TLRs). Since the role of TLR2 and 4 after traumatic brain injury (TBI) is still unclear, we examined the outcome and the expression of pro-inflammatory mediators after experimental TBI in Tlr2/4-/- and wild-type (WT) mice. Tlr2/4-/- and WT mice were subjected to controlled cortical injury and contusion volume and brain edema formation were assessed 24 h thereafter. Expression of inflammatory markers in brain tissue was measured by quantitative PCR 15 min, 3 h, 6 h, 12 h, and 24 h after controlled cortical impact (CCI). Contusion volume was significantly attenuated in Tlr2/4-/- mice (29.7 ± 0.7 mm3 as compared to 33.5 ± 0.8 mm3 in WT; p < 0.05) after CCI while brain edema was not affected. Only interleukin (IL)-1ß gene expression was increased after CCI in the Tlr2/4-/- relative to WT mice. Inducible nitric oxide synthetase, TNF, IL-6, and COX-2 were similar in injured WT and Tlr2/4-/- mice, while the increase in high-mobility group box 1 was attenuated at 6 h. TLR2 and 4 are consequently shown to potentially promote secondary brain injury after experimental CCI via neuroinflammation and may therefore represent a novel therapeutic target for the treatment of TBI.

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 mug/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. NFkappaB 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 4 modulates myocardial ischaemia-reperfusion injury: Role of matrix metalloproteinases.

UNLABELLED: Toll-like receptor 4 (TLR4) mediates innate immune responses following endotoxemia and myocardial ischaemia-reperfusion (I/R) injury. Pre-treatment with the major TLR4 ligand lipopolysaccharide (LPS) reduces infarct size. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play a crucial role in endotoxemia possibly also determining I/R injury. AIMS: We investigated the influence of TLR4 on infarct size and assessed the influence of MMP and TIMP regulation on I/R injury. METHODS: Left anterior descending artery (LAD) occlusion was performed on wild-type (C3H/HeN) and TLR4-deficient (C3H/HeJ) mice. Animals were stimulated with LPS (1 mg/kg) or PBS 16 h ahead of 60 min LAD ligation. After 24 h of reperfusion, triphenyltetrazolium chloride staining was performed and infarct size was measured by planimetry. MMP- and TIMP-mRNA expression were determined by RPA after 3 h of reperfusion. MMP zymographic activity was monitored 6 h after occlusion. RESULTS: TLR4-deficient mice and LPS-treated wild-type mice showed significantly reduced infarct areas. LPS-stimulation significantly increased the overall MMP/TIMP mRNA expression ratio due to elevated MMP-3, -8, -9, and TIMP-1 in wild-type mice. I/R overall reduced the MMP/TIMP ratio due to increased MMP-1, TIMP-1, and -3 mRNA expression. CONCLUSIONS: LPS pre-treatment and TLR4-deficiency led to a decreased infarct size. However, infarct area and MMP/TIMP ratio were not correlated. This means that in TLR4-deficient mice MMP/TIMP ratios are not determining the infarct size.

Myotrophin/V-1 does not act as an extracellular signal to induce myocyte hypertrophy.

The myotrophin/V-1 protein was originally found to be elevated in failing heart tissues and was described as an exogenously acting hypertrophy-inducing factor. However, several studies have proposed only intracellular functions for this protein. We investigated whether this protein is an exogenously acting hypertrophy-inducing trophin or an intracellular nuclear factor of kappa B (NFkappaB) regulatory protein. In the current report, immunofluorescence and cell fractionation studies showed that myotrophin is present only in the cytoplasm and is not actively released into the extracellular environment in response to hypertrophy-inducing stimuli. Moreover, in response to ischemia/reperfusion injury, an active release of myotrophin from adult rat myocardium was not observed. Furthermore, protein synthesis studies in rat neonatal myocytes indicated that exogenous myotrophin did not induce hypertrophy. On the other hand, myotrophin stimulates the generation of NFkappaB dimers in vitro and thus regulates the NFkappaB-mediated transcription in cardiac myocytes. Taken together, these studies suggest that myotrophin is a strictly cytosolic protein that regulates the NFkappaB-mediated transcriptional process.

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-1beta, 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-kappaB, 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.

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)-1beta, 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-6Ralpha 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-kappaB 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-1beta (IL-1beta), 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-1beta mRNA and protein expression in wild-type mice. In contrast, mRNA and protein levels for TNF and IL-1beta 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-kappaB 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/dt(max). 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.

Nuclear factor-kappaB 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 kappaB (NF-kappaB) and given that NF-kappaB mediates cytoprotective responses, we asked whether NF-kappaB 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 IkappaBalpha protein (IkappaBalphaDeltaN) that prevents nuclear translocation of NF-kappaB in cardiac myocytes. Triphenyltetrazolium chloride staining showed that infarct size was approximately 50% greater (P<0.02) in the IkappaBalphaDeltaN mice compared with littermate controls at 24 hours. The prevalence of cardiac myocyte apoptosis was significantly greater (P<0.008) in the IkappaBalphaDeltaN 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 IkappaBalphaDeltaN 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-kappaB have increased susceptibility to tissue injury after acute left anterior descending occlusion. These studies suggest that the cytoprotective effects of NF-kappaB are mediated, at least in part, by Bcl-2 or c-IAP1.