Myocardial TNF-α Research Articles

Overexpression of Hsp20 prevents endotoxin-induced myocardial dysfunction and apoptosis via inhibition of NF-κB activation

The occurrence of cardiovascular dysfunction in sepsis is associated with a significantly increased mortality rate of 70% to 90% compared with 20% in septic patients without cardiovascular impairment. Thus, rectification or blockade of myocardial depressant factors should partly ameliorate sepsis progression. Heat shock protein 20 (Hsp20) has been shown to enhance myocardial contractile function and protect against doxorubicin-induced cardiotoxicity. To investigate the possible role of Hsp20 in sepsis-mediated cardiac injury, we first examined the expression profiles of five major Hsps in response to lipopolysaccharide (LPS) challenge, and observed that only the expression of Hsp20 was downregulated in LPS-treated myocardium, suggesting that this decrease might be one of the mechanisms contributing to LPS-induced cardiovascular defects. Further studies using loss-of-function and gain-of-function approaches in adult rat cardiomyocytes verified that reduced Hsp20 levels were indeed correlated with the impaired contractile function. In fact, overexpression of Hsp20 significantly enhanced cardiomyocyte contractility upon LPS treatment. Moreover, after administration of LPS (25 μg/g) in vivo, Hsp20 transgenic mice (10-fold overexpression) displayed: 1) an improvement in myocardial function; 2) reduced the degree of cardiac apoptosis; and 3) decreased NF-κB activity, accompanied with reduced myocardial cytokines IL-1β and TNF-α production, compared to the LPS-treated non-transgenic littermate controls. Thus, the increases in Hsp20 levels can protect against LPS-induced cardiac apoptosis and dysfunction, associated with inhibition of NF-κB activity, suggesting that Hsp20 may be a new therapeutic agent for the treatment of sepsis.

Improvement in cardiac function after bone marrow cell thearpy is associated with an increase in myocardial inflammation

The mechanisms for the beneficial impact of bone marrow cell (BMC) therapy after myocardial infarction (MI) are ill defined. We hypothesized that the implanted cells improve function by attenuating post-MI inflammation and repair. In mice, 3 x 10(5) fresh BMCs were implanted immediately after coronary ligation. Cardiac function was evaluated over time. Inflammatory cytokines and cells were measured, and their impacts on the (myo)fibroblastic repair response, angiogenesis, and scar formation were determined. All differences below had P values of <0.05. BMC implantation reduced the decline in fractional shortening and ventricular dilation. Invasive hemodynamics confirmed a difference in systolic function at day 7 and diastolic function at day 28 favoring the BMC group. Interestingly, BMC implantation caused a 1.6-fold increase in the number of macrophages infiltrating the infarct but did not affect neutrophils. This increase was associated with a 1.9-fold higher myocardial TNF-alpha level. The heightened inflammatory response was associated with a 1.4-fold induction of transforming growth factor-beta and a 1.3-fold induction of basic fibroblast growth factor. These changes resulted in a 1.6-fold increase in alpha-smooth muscle actin and a 1.9-fold increase in total discoidin domain receptor 2-expressing cells in the BMC group. These two markers are expressed by cardiac (myo)fibroblasts. Capillary density in the border zone increased 2.0-fold. Consistent with a more robust repair-mediated scar "contracture," the final scar size was 0.7-fold smaller in the BMC group. In conclusion, after MI, BMC therapy induced a more robust inflammatory response that improved the "priming" of the (myo)fibroblast repair phase. Enhancing this response may further improve the beneficial impact of cellular therapy.

Abstract 3212: Dietary-Induced Insulin Resistance Associated with Dyslipidemia Induces Progressive Cardiac Dysfunction in Rats as Evidenced by Echocardiography

Background: A major complication of diabetes is the development of cardiac dysfunction in absence of vascular disease. Metabolic disorders such as insulin resistance (IR) and dyslipidemia (DL) might contribute to the induction of diabetic cardiomyopathy (DCM). However, few relevant animal models are currently available for studying the time-course of DCM and evaluating experimental therapeutics. We developed a rodent model of dietary-induced IR combined or not with DL in order to investigate the impact of chronic IR and DL on in vivo myocardial function. Methods &amp; Results: Male Sprague-Dawley rats were fed a western-type diet (65% fat; 15% fructose; WD: n=12). DL was induced by combining the western diet with i.p . injections of a nonionic surface-active agent (P-407; 0.2 mg/kg, 3 times/wk; WD-P407 n=9). A chow diet was used as control (Chow: n=9). At 6, 11 and 14 wks, cardiac function was assessed by echocardiography. After 6 wks, plasma insulin was significantly increased in both WD and WD-P407 groups ( P&lt; 0.05 vs. Chow). Fasting blood glucose increased in WD group while plasma lipids markedly accumulated in WD-P407-treated rats ( P&lt; 0.05 and P&lt; 0.01 vs. Chow, respectively). Pulse-wave Doppler indicated impaired diastolic function at 14 wks (E/A wave ratio: WD-P407: 1.42±0.06 vs. Chow: 1.65±0.11). M-mode imaging showed no significant differences in cardiac function and geometry under basal conditions. However, fractional shortening (FS) was significantly depressed under dobutamine stress in WD group at 14 wks (FS in % of baseline: 151±9% vs 196±7%; P&lt; 0.05) whereas systolic dysfunction appeared as early as 11 wks and worsened at 14 wks in WD-P407 animals ( P&lt; 0.05 and P&lt; 0.01 vs. Chow, respectively). Finally, compared to Chow, myocardial lipid tissue content and pro-inflammatory cytokine TNF-α were significantly higher in WD and WD-P407 groups, the cardiac lipid accumulation being more pronounced in the later. Conclusions: DL exacerbated cardiac lipotoxicity and functional complications associated with IR. This experimental model of combined IR and DL closely mimics the main clinical manifestations of DCM and might therefore constitute a useful tool for the evaluation of pharmacological treatments.

Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats

Mast cells have diverse roles throughout the body as evidenced by their heterogeneous nature. In the heart, cardiac mast cells have been implicated in left ventricular (LV) remodeling in response to elevated myocardial stress. Accordingly, the purpose of this study was to use mast cell deficient rats (Ws/Ws) to delineate the interaction between cardiac mast cell activation and LV remodeling. LV matrix metalloproteinase (MMP) activity, fibrillar collagen, TNF-α levels, and LV diameter were compared in Ws/Ws and wild type (WT) rats subjected to 5 d ( n = 3/group) and 8 weeks ( n = 4/group) of aortocaval fistula-induced volume overload. In contrast to attenuation of myocardial remodeling in the Ws/Ws group: 1) MMP-2 activity was significantly increased in the WT group at 5 days; 2) there was marked degradation of the extracellular collagen matrix in WT at 5 days and 8 weeks; 3) the percent increase in LV diameter from baseline was significantly greater in WT at 2, 4, 6, and 8 weeks post-fistula; and 4) myocardial TNF-α levels were markedly elevated in the WT group at 5 days post-fistula. These results underscore the importance of cardiac mast cells in mediating MMP activation, collagen degradation and LV dilatation and suggest that mast cell-derived TNF-α plays a role in early myocardial remodeling.

Antigenic stimulation in the simian model of HIV infection yields dilated cardiomyopathy through effects of TNFα

To investigate a role for endogenous myocardial cytokine production in the development of HIV-associated cardiomyopathy. Cardiomyopathy is a late-stage sequela of HIV infection. Although pathogenesis of this condition in HIV infection is poorly defined, inflammatory cytokines are recognized for their detrimental effects on myocardial structure and function. HIV infection is characterized by chronic immune activation and inflammatory cytokine dysregulation. As the myocardium itself is a rich potential source of inflammatory cytokines, HIV-mediated cytokine dysregulation may be an important contributor to development of HIV cardiomyopathy. An antigenic stimulation protocol conducted in the simian immunodeficiency virus (SIV) model of HIV infection was used to study the effects of endogenous cytokine production on myocardial structure and function. Twenty-six rhesus monkeys were assigned to treatment groups for a 35-day study. Animals were SIV-infected; SIV-infected and treated with killed Mycobacterium avium complex bacteria (MAC); SIV-infected, MAC-treated, and given the TNFalpha antagonist etanercept; or uninfected and MAC-treated. All animals were subjected to weekly echocardiographic studies. Hearts were collected for further evaluation at euthanasia. SIV-infected, MAC-treated animals developed significant systolic dysfunction [left ventricular ejection fraction (LVEF) decline of 19 +/- 2%] and ventricular chamber dilatation [left ventricular end-diastolic diameter (LVEDD) increase of 26 +/- 6%] not seen in other groups. Concurrent treatment with etanercept prevented development of these changes, implicating a causative role of myocardial TNFalpha. SIV-infected animals develop exaggerated myocardial pathology on stimulation with the ubiquitous environmental agent MAC. These responses are TNFalpha-dependent and may play a significant role in the development of cardiomyopathy in HIV infection.

Positive Correlation of Tumor Necrosis Factor-α Early Expression in Myocardium and Ventricular Arrhythmias in Rats with Acute Myocardial Infarction

Tumor necrosis factor-alpha (TNF-alpha) is found to play important roles in acute myocardial infarction (AMI). Ventricular arrhythmias arising from AMI are leading causes of sudden cardiac death (SCD). We sought to clarify the effect of TNF-alpha early expression on ventricular arrhythmias in rats with AMI and its mechanism. Rats with AMI were induced by left anterior descending coronary branch ligation. The mRNA and protein levels of TNF-alpha in myocardium were detected by real-time fluorescent quantitative PCR, Western blotting and histochemistry. Meanwhile, electrocardiogram was recorded. Different concentrations of TNF-alpha were added to isolated rat hearts in isolated heart perfusions. Effect of TNF-alpha on intracellular Ca(2+) concentration was detected by laser confocal technique. In AMI rats, mRNA and protein levels of TNF-alpha were higher than control (p <0.05), and the occurrence time of ventricular arrhythmias coincided with the secretion of TNF-alpha. TNF-alpha may cause ventricular arrhythmias in isolated rat heart perfusion models. Intracellular Ca(2+) intensity may quickly be increased by TNF-alpha. Our results reveal the positive correlation between TNF-alpha early expression and ventricular arrhythmias in rats with AMI. This effect may be associated with the increased intracellular Ca(2+) intensity caused by TNF-alpha.

Prospective clinical and experimental studies on the cardioprotective effect of ulinastatin following severe burns

Objective To investigate the preventive effect of ulinastatin on shock in the heart after burn. Methods In an open prospective clinical study 34 adults with burns >50% total body surface area were randomly divided into control (B) and ulinastatin-treated (U) groups. All underwent routine treatment, and group U received 100,000 U ulinastatin intravenously three times a day for 1 week. In an animal experiment, 72 healthy rats underwent equivalent burn, similar division into groups B and U, and resuscitation according to Parkland's formula. Rats in group U received ulinastatin (40,000 U/kg) immediately after burn. Myocardial pathomorphology, plasma cTnI, CK-MB and PMNE, myocardial MDA, TNF-α, IL-10 and caspase-3 activity and cardiocyte apoptosis were determined. Results Plasma cTnI, CK-MB, and PMNE were higher in clinical group B than group U. In the animal experiment, plasma cTnI, CK-MB, myocardial MDA, TNF-α, IL-10 and caspase-3 activity, and apoptotic index and myocardial pathomorphological changes were significantly less in group U than in group B, save IL-10. Conclusion The clinical and experimental data showed that ulinastatin relieved myocardial damage from severe burn. The mechanism might involve modulation of the anti- and pro-inflammatory balance and lipid peroxidation, and decreased myocardiocyte apoptosis.

Candesartan improves myocardial damage in obese mice with viral myocarditis and induces cardiac adiponectin

Purpose To clarify the mechanism of the effects of angiotensin II receptor type 1 antagonist, candesartan, upon cardiac adiponectin in the combination of myocarditis with obesity, we examined obese KKAy mice with acute viral myocarditis treated by candesartan and investigated cardiac adiponectin regulation. Methods Mice were divided into candesartan early treatment group (Can-early) receiving orally candesartan at daily dose of 10 mg/kg 7 days starting before viral inoculation and then 7 days; candesartan late treatment group (Can-late) or vehicle (Vehicle) receiving candesartan starting simultaneously with viral inoculation and then 7 days. Encephalomyocarditis virus was used to induce the acute viral myocarditis. Differences in myocardial damages, serum adiponectin and myocardial expression of adiponectin, tumor necrosis factor-α (TNF-α), CCAAT/enhancer binding proteinα (C/EBPα) and peroxisome proliferator-activated receptor γ (PPAR-γ) and nuclear factor-κB (NF-κB) mRNA among three groups were determined on days 0, 4 and 7 after viral inoculation. Results Mice in Can-early and Can-late groups showed reduced myocardial necrosis and cellular infiltration as compared with those in the Vehicle. On day 4 the circulating adiponectin levels were significantly higher in Can-early than those in Vehicle. Mice in Vehicle had significantly reduced in myocardial adiponectin mRNA after viral myocarditis. Cardiac adiponectin mRNA was significantly higher in Can-early and in Can-late than in Vehicle on days 4 and 7. Cardiac C/EBPα in Can-early and Can-early groups was significantly increased on day 4. Myocardial NF-κB and TNF-α mRNA in Can-early and Can-late groups were significantly reduced on day 7. Conclusion Candesartan treatment improved myocardial injury in obese mice with acute viral myocarditis and induced expression of cardiac adiponectin with the induction of C/EBPα as well as the reduction of cardiac NF-κB and TNF-α.

Cardioplegic ischemia or reperfusion: Which is a main trigger for tumor necrosis factor production?

Background Tumor necrosis factor alpha (TNF-α) is a key cytokine in the pathogenesis of ischemia–reperfusion injury (I/R) that also possesses negative inotropic and direct cardiotoxic effects. We investigated whether myocardial ischemia and/or reperfusion is the trigger for TNF-α synthesis and whether TNF-α release is time dependent. Methods Isolated rat hearts undergoing 30 min of coronary perfusion with modified Krebs–Henseleit solution followed by cardioplegic arrest for 60 min of global cardioplegic normothermic ischemia (GCI) and 30 min of reperfusion using a modified Langendorff model. Myocardial TNF-mRNA expression and TNF-α protein levels in effluent from the coronary sinus were measured at baseline and then after 15, 30, and 60 min of GCI and after 10 and 30 min of reperfusion. Results GCI induced myocardial TNF-α mRNA expression and elevation protein TNF-α levels in a time-dependent manner after 30 min of ischemia from 78 ± 17 pg/ml to 915 ± 287 pg/ml after 60 min ( p < 0.0015). Reperfusion did not cause time-dependent increase of TNF-α synthesis and release but was accompanied by progressive decrease of left ventricular (LV) function. There was a correlation between TNF-α protein levels and depression of LV function immediately after GCI but not with TNF-α protein levels at 30 min of reperfusion. Conclusion This study demonstrated that myocardial ischemia rather than reperfusion is the main trigger for time-dependent TNF-α synthesis. Depression of LV function during reperfusion correlated significantly only with TNF-α levels at the end of GCI.

Signalling mechanisms and phospholipase a2 translocation in TNF-α mediated cytoprotection in ischaemia

Tumor Necrosis Factor-α (TNF-α) and the cytosolic phospholipase A2 (cPLA2) are involved in the cellular inflammatory response and ischaemic injury. During myocardial ischaemia TNF-α is endogenously generated, activating cPLA2. In addition, low dose TNF-α confers cytoprotection. cPLA2 activation results in the generation of mediators of cellular injury, apoptosis and necrosis. We have therefore investigated the role of cPLA2 in TNF-α mediated cytoprotection. Murine C2C12 myoblasts were grown to 70% confluency and differentiated into myotubes.

Prevention of TNFα-associated myocardial dysfunction resulting from cardiopulmonary bypass and cardioplegic arrest by glucocorticoid treatment

Cardiac surgery on cardiopulmonary bypass (CPB) results in progressive myocardial dysfunction, despite unimpaired coronary blood flow, and is associated with increased myocardial tumor necrosis factor-alpha (TNFalpha) expression. We investigated whether anti-inflammatory treatment prevents increased TNFalpha expression and myocardial dysfunction after CPB. Baseline systemic hemodynamics, myocardial contractile function, aortic and coronary blood flow were measured in anesthetized pigs. Then, placebo (PLA; saline; n=7) or methylprednisolone (MP; 30 mg/kg; n=6) was infused intravenously and CPB was instituted. Global ischemia was induced for 10 min by aortic cross-clamping, followed by 1 h of cardioplegic cardiac arrest. After declamping and reperfusion, CPB was terminated after a total of 3 h. Measurements were repeated at 15 min, 4 h, and 8 h following termination of CPB. Systemic TNFalpha-plasma concentrations and left ventricular TNFalpha expression were analyzed. With unchanged coronary blood flow in both groups, a progressive loss of myocardial contractile function to 38+/-2% of baseline (p<0.01) and cardiac index to 48+/-6% of baseline (p<0.01) at 8 h after CPB in PLA was attenuated in MP (myocardial function: 72+/-3%, p<0.01 vs PLA; cardiac index: 78+/-6%, p<0.05 vs PLA). Systemic TNFalpha was increased at 8 h in PLA compared to MP (243+/-34 vs 90+/-34 pg/ml, p<0.05). Myocardial TNFalpha was increased at 8 h after CPB compared to baseline and MP (p<0.05). Myocardial TNFalpha immunostaining was more pronounced in PLA than in MP (p<0.05), with TNFalpha-mRNA localization predominantly to cardiomyocytes. Methylprednisolone attenuates both systemic and myocardial TNFalpha increases and progressive myocardial dysfunction induced by cardiac surgery, suggesting a key role for TNFalpha.

Growth hormone- and pressure overload-induced cardiac hypertrophy evoke different responses to ischemia-reperfusion and mechanical stretch

Objective. To compare the molecular, histological, and functional characteristics of growth hormone (GH)- and pressure overload-induced cardiac hypertrophy, and their responses to ischemia-reperfusion and mechanical stretch. Design. Four groups of male Wistar rats were studied: aortic banding ( n = 24, AB) or sham ( n = 24, controls) for 10 weeks, and GH treatment ( n = 24; 3.5 mg/kg/day, GH) or placebo ( n = 24, controls) for 4 weeks. At 13 weeks, the rats were randomly subjected to: (i) assessment of basal left ventricular mRNA expression of sarcoplasmic reticulum calcium-ATPase (SERCA-2), phospholamban (PLB), and Na +–Ca 2+ exchanger (NCX) and collagen volume fraction (CVF) ( Protocol A, 8 rats in each group); (ii) left ventricular no-flow ischemia with simultaneous evaluation of intracellular Ca 2+ handling and ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) content ( Protocol B, 12 rats in each group); or (iii) left ventricular mechanical stretch for 40 min with assessment of tumor necrosis-α (TNF-α) mRNA ( Protocol C, 4 rats in each group). Protocol B and C were carried out in a Langendorff apparatus. Results. In Protocol A, no difference was found as to myocardial mRNA content of Ca 2+ regulating proteins and CVF in GH animals vs controls. In contrast, in the AB group, myocardial mRNA expression of SERCA-2 and PLB was downregulated while that of NCX and CVF were increased vs. controls ( p < 0.05). In Protocol B, recovery of left ventricular function was significantly decreased in AB vs GH groups and controls and this was associated with 1.6-fold increase in intracellular Ca 2+ overload during reperfusion ( p < 0.05). Baseline ATP content was similar in the four study groups, whereas PCr and Pi was lower in AB vs GH rats and controls. However, the time courses of high-energy phosphate metabolic changes did not differ during ischemia and reperfusion in the four study groups. In Protocol C, no detectable TNF-α mRNA level was found in the left ventricular myocardium of GH treated rats and controls at baseline, while a modest expression was noted in AB animals. Mechanical stretch resulted in de novo myocardial TNF-α mRNA expression in GH group and controls, which was dramatically increased in AB animals (≈5-fold above baseline, p < 0.001). Conclusions. The data show that cardiac hypertrophy activated by short-term GH treatment confers cardioprotection compared with pressure overload with regard to molecular and histological characteristics, and responses to ischemia-reperfusion and mechanical stretch.

Mechanisms of Cardiac Depression Caused by Lipoteichoic Acids From Staphylococcus aureus in Isolated Rat Hearts

Lipoteichoic acid (LTA) represents a major virulence factor in gram-positive sepsis. In the present study we perfused isolated rat hearts for 180 minutes with highly purified LTA from Staphylococcus aureus. A progressive decline of left ventricular contractile function paralleled by the expression of myocardial tumor necrosis factor-alpha (TNF-alpha) mRNA and protein as well as the release of TNF-alpha into the perfusate was observed in LTA-perfused hearts. Employment of an anti-TNF-alpha antibody completely prevented the loss in contractile function. When CD14, a prominent pathogen recognition receptor, was blocked by a specific antibody, induction of TNF-alpha mRNA and protein release as well as the associated cardiodepression was diminished in response to LTA. Synthesis of TNF-alpha protein was located to interstitial cells of LTA-challenged hearts as detected by immunohistochemistry. Besides progressive cardiodepression, coronary perfusion pressure (CPP) was moderately increased in LTA-perfused hearts. This was accompanied by the release of thromboxane A2 (TXA2) into the perfusate and the induction of cyclooxygenase (Cox)-2 mRNA and protein in the myocardium. Blocking of TXA2 by the nonspecific Cox inhibitor indomethacin, the thromboxane receptor antagonist daltroban, or the selective Cox-2 inhibitor NS-398 prevented the increase in CPP. LTA causes cardiac depression by activating myocardial TNF-alpha synthesis via CD14 and induces coronary vascular disturbances by activating Cox-2-dependent TXA2 synthesis. These phenomena may contribute to cardiac depression in gram-positive sepsis.

Inhibitor-κB kinase-β regulates LPS-induced TNF-α production in cardiac myocytes through modulation of NF-κB p65 subunit phosphorylation

TNF-α is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-κB family of transcription factors are essential regulators of myocardial TNF-α gene expression, recent developments in our understanding of the modulation of NF-κB activity through posttranslational modification of NF-κB subunits suggest that the present view of NF-κB-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-α production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-α production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser536, a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-β reduced LPS-induced TNF-α production 38-fold, TNF-α mRNA levels 6-fold, and IκB-α phosphorylation 5-fold and degraded IκB-α 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-α production 3.5-fold, whereas overexpression of dominant-negative IKK-β reduced LPS-induced TNF-α production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-α had no effect on p65 phosphorylation or TNF-α production, revealing that IKK-β, not IKK-α, plays a central role in regulation of p65 phosphorylation at Ser536and TNF-α production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-α gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-κB signaling in TNF-α gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction.

Selective disruption of MMP-2 gene exacerbates myocardial inflammation and dysfunction in mice with cytokine-induced cardiomyopathy

Tumor necrosis factor-alpha (TNF-alpha) plays a pathophysiological role in the development and progression of heart failure. Matrix metalloproteinase (MMP)-2 is involved in extracellular matrix remodeling. Recent evidence suggests a protective role for this protease against tissue inflammation. Although MMP-2 is upregulated in the failing heart, little is known about its pathophysiological role. We thus hypothesized that ablation of the MMP-2 gene could affect cardiac remodeling and failure in TNF-alpha-induced cardiomyopathy. We crossed transgenic mice with cardiac-specific overexpression of TNF-alpha (TG) with MMP-2 knockout (KO) mice. Four groups of male and female mice were studied: wild-type (WT) with wild MMP-2 (WT/MMP(+/+)), WT with MMP-2 KO (WT/MMP(-/-)), TNF-alpha TG with wild MMP-2 (TG/MMP(+/+)), and TG with MMP-2 KO (TG/MMP(-/-)). The upregulation of MMP-2 zymographic activity in TG/MMP(+/+) mice was completely abolished in TG/MMP(-/-) mice, and other MMPs and tissue inhibitors of metalloproteinase were comparable between groups. Survival was shorter for male TG/MMP(-/-) than TG/MMP(+/+) mice. Female TG/MMP(-/-) mice were more severely affected than TG/MMP(+/+) mice with diminished cardiac function. Myocardial TNF-alpha and other proinflammatory cytokines were increased in TG/MMP(+/+) mice, and this increase was similarly observed in TG/MMP(-/-) mice. The extent of myocardial infiltrating cells including macrophages was greater in TG/MMP(-/-) than in TG/MMP(+/+) mice. Selective ablation of the MMP-2 gene reduces survival and exacerbates cardiac failure in association with the increased level of myocardial inflammation. MMP-2 may play a cardioprotective role in the pathogenesis of cytokine-induced cardiomyopathy.

Pivotal Role of gp91 phox -Containing NADH Oxidase in Lipopolysaccharide-Induced Tumor Necrosis Factor-α Expression and Myocardial Depression

Lipopolysaccharide (LPS) induces cardiomyocyte tumor necrosis factor-alpha (TNF-alpha) production, which is responsible for myocardial depression during sepsis. The aim of this study was to investigate the role of gp91phox-containing NADH oxidase signaling in cardiomyocyte TNF-alpha expression and myocardial dysfunction induced by LPS. In cultured mouse neonatal cardiomyocytes, LPS increased NADH oxidase (gp91phox subunit) expression and superoxide generation. Deficiency of gp91phox or inhibition of NADH oxidase blocked TNF-alpha expression stimulated by LPS. TNF-alpha induction was also inhibited by tempol, N-acetylcysteine, or 1,3-dimethyl-2-thiourea. NADH oxidase activation by LPS increased ERK1/2 and p38 phosphorylation, and inhibition of ERK1/2 and p38 phosphorylation blocked the effect of NADH oxidase on TNF-alpha expression. Isolated mouse hearts were perfused with LPS (5 microg/mL) alone or in the presence of apocynin for 1 hour. Myocardial TNF-alpha production was decreased in gp91phox-deficient or apocynin-treated hearts compared with those of wild type (P<0.05). To investigate the role of gp91phox-containing NADH oxidase in endotoxemia, mice were treated with LPS (4 mg/kg IP) for 4 and 24 hours, and their heart function was measured with a Langendorff system. Deficiency of gp91phox significantly attenuated LPS-induced myocardial depression (P<0.05). gp91phox-Containing NADH oxidase is pivotal in LPS-induced TNF-alpha expression and cardiac depression. Effects of NADH oxidase activation are mediated by ERK1/2 and p38 MAPK pathway. The present results suggest that gp91phox-containing NADH oxidase may represent a potential therapeutic target for myocardial dysfunction in sepsis.

Simvastatin decreases myocardial tumor necrosis factor α content in heart transplant recipients

Background Statins improve patient survival and decrease rejection episodes in heart transplant recipients. We studied the effects of simvastatin treatment on myocardial tumor necrosis factor α (TNF-α) expression; TNF-α is a potent pro-inflammatory cytokine associated with hypertrophy and fibrosis in heart transplant recipients. Methods We randomized 10 consecutive heart transplant recipients to receive either 20 mg/day simvastatin ( n = 5) or placebo ( n = 5) for 6 months after cardiac transplantation. Routine surveillance endomyocardial biopsy specimens were obtained from all patients. We analyzed tissues for myocardial TNF-α content, total collagen content, and myocyte size using semiquantitative immunohistochemistry. Results Myocyte size and total collagen content of placebo and simvastatin groups did not show a statistically significant difference at any biopsy time point. Myocardium TNF-α content (% tissue area stained) at 1 week after transplantation was similar in the simvastatin and placebo groups. At the 24 th week after transplantation, when compared with Week 1 values, we found a significant decrease in myocardium TNF-α content in the simvastatin group (15.0% ± 2.3% vs 5.8% ± 2.4%, p = 0.02) that was not observed in the placebo group (15.0% ± 1.5% vs 12.0% ± 2.6%, p = not significant). Conclusion Simvastatin treatment in heart transplant recipients decreased myocardium TNF-α expression. This decrease did not translate into a difference in the markers of hypertrophy. However, decreased myocardial TNF-α may be a marker of a general statin-mediated decrease in inflammation in the transplanted heart that leads to improved graft and patient survival.

Myocardial protective effect of FR167653; a novel cytokine inhibitor in ischemic-reperfused rat heart

In this study, a newly synthesized cytokine inhibitor FR167653 was investigated using a rat heart ischemia-reperfusion model to prove its myocardial protective effect and its role in the inhibition of cytokine production in ischemic myocardium. Studies were performed with isolated, Langendorff-perfused Lewis rat hearts (n=80) which were either treated with FR167653 or untreated, as the control group, and subjected to ischemia-reperfusion. Reperfusion followed by 30min of 37 degrees C ischemia induced marked myocardial cytokine expression and activated p38MAPK. FR167653 administered before ischemia and during reperfusion significantly reduced ischemia-activated myocardial TNFalpha mRNA expression (190+/-97 vs. 4805+/-3017, P=0.024) as well as TNFalpha production (0 vs. 9.6+/-2.5 ng/ml, P<0.05) and also inhibited p38 MAPK activation. Its administration improved recovery of cardiac contractile function during reperfusion: LVDP (130+/-18 vs. 82+/-21 mmHg (P=0.002)), max/min dP/dt (2812+/-328/-2283+/-216 vs. 1520+/-424/-1325+/-237 mmHg/s, P=0.003). CPK leakage was significantly reduced in FR167653 treated hearts versus untreated hearts (54+/-6 vs. 0.5+/-0.1, P<0.05) and reduction of coronary flow was improved (110+/-13 vs. 77+/-11%) 1h after beginning of reperfusion (P<0.05). Moreover, FR administration attenuated the number of TUNEL positive cardiomyocytes (3+/-1 vs. 9+/-2%). These data demonstrated positive inotropic and antiapoptotic effects of a newly synthesized compound (FR167653) of cytokine inhibitors and its inhibitory effect on myocardial TNFalpha production and p38 MAPK activation in ischemic-reperfused rat heart. This suggested that cytokine inhibition is significant as a method for myocardial protection against ischemia-reperfusion injury.

Signaling for myocardial depression in hemorrhagic shock: roles of Toll-like receptor 4 and p55 TNF-α receptor

Hemorrhagic shock causes myocardial contractile depression. Although this myocardial disorder is associated with increased expression of tumor necrosis factor-alpha (TNF-alpha), the role of TNF-alpha as a myocardial depressant factor in hemorrhagic shock remains to be determined. Moreover, it is unclear which TNF-alpha receptor mediates the myocardial depressive effects of TNF-alpha. Toll-like receptor 4 (TLR4) regulates cellular expression of proinflammatory mediators following lipopolysaccharide stimulation and may be involved in the tissue inflammatory response to injury. The contribution of TLR4 signaling to tissue TNF-alpha response to hemorrhagic shock and TLR4's role in myocardial depression during hemorrhagic shock are presently unknown. We examined the relationship of TNF-alpha production to myocardial depression in a mouse model of nonresuscitated hemorrhagic shock, assessed the influence of TLR4 mutation, resulting in defective signaling, on TNF-alpha production and myocardial depression, and determined the roles of TNF-alpha and TNF-alpha receptors in myocardial depression using a gene knockout (KO) approach. Hemorrhagic shock resulted in increased plasma and myocardial TNF-alpha (4.9- and 4.5-fold, respectively) at 30 min and induced myocardial contractile depression at 4 h. TLR4 mutation abolished the TNF-alpha response and attenuated myocardial depression (left ventricular developed pressure of 43.0 +/- 6.2 mmHg in TLR4 mutant vs. 30.0 +/- 3.6 mmHg in wild type, P < 0.05). TNF-alpha KO also attenuated myocardial depression in hemorrhagic shock, and the p55 receptor KO, but not the p75 receptor KO, mimicked the effect of TNF-alpha KO. The results suggest that TLR4 plays a novel role in signaling to the TNF-alpha response during hemorrhagic shock and that TNF-alpha through the p55 receptor activates a pathway leading to myocardial depression. Thus TLR4 and the p55 TNF-alpha receptor represent therapeutic targets for preservation of cardiac mechanical function during hemorrhagic shock.

Increased toll-like receptor 4 in the myocardium of patients requiring left ventricular assist devices

Background Cytokine activation in the myocardium of deteriorating patients with heart failure who undergo left ventricular assist-device (LVAD) implantation has been documented, but the underlying mechanisms remain poorly understood. We hypothesized the innate immune system is activated with expression of Toll-like receptor 4 (TLR4), leading to cytokine activation in these patients. Methods We used quantitative real-time reverse-transcriptase polymerase chain reaction to measure TLR4, interleukin-1 (IL-1) receptor, IL-1β, IL-6, and tumor necrosis factor α (TNF-α) mRNA expression in myocardial samples from 36 patients. We compared 18 patients who underwent LVAD implantation with 18 patients with less severe heart failure who underwent elective heart transplantation. Results Toll-like receptor 4 expression was 1.69-fold greater ( p < 0.05) and IL-1 receptor expression was 3.64–fold greater ( p < 0.0001) in the deteriorating patients who required LVADs. Myocardial TNF-α (1.71-fold, p < 0.05), IL-6 (2.57-fold, p < 0.005), and IL-1β (9.78-fold, p < 0.001) also were increased in the LVAD candidates. Toll-like receptor 4 expression correlated strongly with IL-1 receptor expression ( r= 0.75, p < 0.0001) and with IL-1 β expression in individual patients ( r = 0.7, p < 0.0001). Interleukin-1 receptor expression also correlated with IL-1β expression ( r = 0.78, p < 0.0001) within patients. We found no correlation between TLR4 and either TNF-α or IL-6 expression. Conclusions Patients who required LVAD support showed evidence of innate immune system activation, indicated by an increase in the key effector molecule TLR4 associated with a specific pattern of cytokine expression in the myocardium.