Gregory L. Freeman

β-Adrenergic Blockade in Developing Heart Failure Effects on Myocardial Inflammatory Cytokines, Nitric Oxide, and Remodeling

Background—Whether β-adrenergic blockade modulates myocardial expression of inflammatory cytokines and nitric oxide (NO) in heart failure is unclear. Methods and Results—We administered oral metoprolol or no therapy to rats for 12 weeks after large myocardial infarction and subsequently examined left ventricular (LV) remodeling; myocardial tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 expression; and NO. In untreated rats, echocardiography revealed significant (P<0.001) LV dilatation and systolic dysfunction compared with sham. Papillary muscle studies revealed isoproterenol hyporesponsiveness to be unaltered by NO synthase (NOS) inhibition. Circulating NO metabolites were undetectable. In noninfarcted myocardium, although inducible NOS (iNOS) mRNA was absent, TNF-α, IL-1β, and IL-6 mRNA and protein were markedly elevated compared with sham (P<0.001), with 2-fold higher expression (P<0.025) of IL-6 compared with TNF-α or IL-1β. Metoprolol administration starting 48 hours after infarction (1...BACKGROUND Whether beta-adrenergic blockade modulates myocardial expression of inflammatory cytokines and nitric oxide (NO) in heart failure is unclear. METHODS AND RESULTS We administered oral metoprolol or no therapy to rats for 12 weeks after large myocardial infarction and subsequently examined left ventricular (LV) remodeling; myocardial tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 expression; and NO. In untreated rats, echocardiography revealed significant (P<0.001) LV dilatation and systolic dysfunction compared with sham. Papillary muscle studies revealed isoproterenol hyporesponsiveness to be unaltered by NO synthase (NOS) inhibition. Circulating NO metabolites were undetectable. In noninfarcted myocardium, although inducible NOS (iNOS) mRNA was absent, TNF-alpha, IL-1beta, and IL-6 mRNA and protein were markedly elevated compared with sham (P<0.001), with 2-fold higher expression (P<0.025) of IL-6 compared with TNF-alpha or IL-1beta. Metoprolol administration starting 48 hours after infarction (1) attenuated (P<0.02) LV dilatation and systolic dysfunction, (2) preserved isoproterenol responsiveness (P<0.025) via NO-independent mechanisms, and (3) reduced myocardial gene expression and protein production of TNF-alpha and IL-1beta (P<0. 025) but not IL-6, which remained high. CONCLUSIONS During heart failure development, adrenergic activation contributes to increased myocardial expression of TNF-alpha and IL-1beta but not IL-6, and one mechanism underlying the beneficial effects of beta-adrenergic blockade may involve attenuation of TNF-alpha and IL-1beta expression independent of iNOS and NO.

Long-Term Healthcare and Cost Outcomes of Disease Management in a Large, Randomized, Community-Based Population With Heart Failure

Background—Because of the prevalence and expense of congestive heart failure (CHF), significant efforts have been made to develop disease management (DM) programs that will improve clinical and financial outcomes. The effectiveness of such programs in a large, heterogeneous population of CHF patients remains unknown. Methods and Results—We randomized 1069 patients (aged 70.9±10.3 years) with systolic (ejection fraction 35±9%) or echocardiographically confirmed diastolic heart failure (HF) to assess telephonic DM over an 18-month period. Data were collected at baseline and at 6-month intervals. Survival analysis was performed by Kaplan-Meier and Cox regression methods. Healthcare utilization was defined after extensive record review, with an attempt to account for all inpatient and outpatient visits, medications, and diagnostic tests. We obtained data on 92% of the patients, from nearly 53 000 health-related encounters. Total cost per patient was defined by adding estimated costs for the observed encounters, excluding the cost of the DM. Kaplan-Meier analysis showed that DM patients had a reduced mortality rate (P=0.037), with DM patients surviving an average of 76 days longer than controls. Subgroup analysis showed that DM had beneficial outcomes in patients with systolic HF (hazard ratio 0.62; P=0.040), which was more pronounced in NYHA classes III and IV. Although improvements in NYHA class were more likely with DM (P<0.001), 6-minute walk data from 217 patients in whom data were available at each visit showed no significant benefit from DM (P=0.08). Total and CHF-related healthcare utilization, including medications, office or emergency department visits, procedures, or hospitalizations, was not decreased by DM. Repeated-measures ANOVA for cost by group showed no significant differences, even in the higher NYHA class groups. Conclusions—Participation in DM resulted in a significant survival benefit, most notably in symptomatic systolic HF patients. Although DM was associated with improved NYHA class, 6-minute walk test results did not improve. Healthcare utilization was not reduced by DM, and it conferred no cost savings. DM in HF results in improved life expectancy but does not improve objective measures of functional capacity and does not reduce cost.

Ischemia-Reperfusion of Rat Myocardium Activates Nuclear Factor-κB and Induces Neutrophil Infiltration Via Lipopolysaccharide-Induced CXC Chemokine

BackgroundMechanisms by which neutrophils are attracted to the myocardium in ischemia/reperfusion are not fully defined. Lipopolysaccharide-induced CXC chemokine (LIX), cytokine-induced neutrophil chemoattractant (KC), and macrophage inflammatory protein-2 (MIP-2) are rodent chemokines with potent neutrophil-chemotactic activity. The goals of the present study were to evaluate the roles of these chemokines in a rat model of ischemia/reperfusion and to examine the mechanisms of chemokine induction by oxidative stress and cytokines in cultured cardiomyocytes. Methods and ResultsMale Wistar-Kyoto rats underwent 45 minutes of ligation of the left anterior descending coronary artery, followed by reperfusion for various periods. Compared with sham-operated controls, myocardium from reperfused animals had higher levels of free radicals, increased neutrophil infiltration evidenced histologically and by elevated myeloperoxidase activity, and increased nuclear factor (NF)-&kgr;B DNA binding activity. Ischemia-reperfusion also induced the expression of interleukin-1&bgr;, tumor necrosis factor (TNF)-&agr;, LIX, KC, and MIP-2 mRNA and protein. LIX expression was localized to resident myocardial cells, whereas KC and MIP-2 were expressed only in infiltrating inflammatory cells. Neutralization of LIX inhibited 79% of neutrophil infiltration into previously ischemic myocardium. In contrast, neutralization of KC and MIP-2 reduced neutrophil infiltration by only 28% and 37%, respectively. In cultured cardiomyocytes, LIX expression was induced by oxidative stress or TNF-&agr; and was blocked by the NF-&kgr;B inhibitor pyrrolidinedithiocarbamate. ConclusionsLIX is expressed by resident myocardial cells during ischemia-reperfusion and is induced in cultured cardiomyocytes by oxidative stress or TNF-&agr; via NF-&kgr;B activation. Although KC and MIP-2 are expressed by inflammatory cells infiltrating the myocardium during reperfusion after ischemia, neutrophil recruitment to reperfused rat myocardium is mainly due to cardiomyocyte expression of LIX.

Induction of nuclear factor κB and activation protein 1 in postischemic myocardium

Ischemia/reperfusion induces nuclear factor κB (NF‐κB) and AP‐1 in rat hearts after 15 min of ischemia followed by reperfusion (R) for various periods of time (15 and 30 min, 1, 2, 3, 6, 12, and 24 h). Low levels of NF‐κB and no signal for AP‐1 were detected in shams and in non‐ischemic tissue distant from the ischemic zone. In postischemic tissue, NF‐κB levels increased biphasically with peak levels at 15 min and again at 3 h R. Immunoblotting showed minimal NF‐κB p50 subunit at all times, with changes in p65 similar to EMSA results. Northern blots showed low p50 and increased p65 expression levels at both 2 and 3 h R. By contrast, AP‐1 increased monophasically, with peak levels at 15 min R, which dropped steadily thereafter. These results indicate that NF‐κB and AP‐1 are differentially regulated during reperfusion, which may be a control mechanism for gene expression in reperfused myocardium.

Tumor Necrosis Factor-α Induces a Biphasic Effect on Myocardial Contractility in Conscious Dogs

Tumor necrosis factor-alpha (TNF-alpha) likely plays a role in the pathophysiology of myocardial depression observed in septic shock. To evaluate the hemodynamic effects of TNF-alpha in vivo while eliminating the influence of altered sympathetic tone, eight conscious chronically instrumented dogs were studied after pretreatment with propranolol (2 mg/kg) and atropine (2mg). Using three sets of piezoelectric crystals to measure left ventricular (LV) volume and LV manometers to measure pressure, we determined load-independent parameters of LV systolic performance before, during, and after infusion of recombinant human TNF-alpha (rhTNF-alpha, 40 micrograms/kg for 1 hour). Plasma was analyze for epinephrine and norepinephrine. Between 1 and 7 hours of exposure, rhTNF-alpha induced significant increases in circulating catecholamines. Norepinephrine rose from 268.6 +/- 47.2 to 426.2 +/- 87.0 pg/mL (P < .05) at 1 hour and peaked at 921.2 +/- 156.8 pg/mL (P < .001) at 4 hours after initiating rhTNF-alpha treatment. Similarly, epinephrine increased from 130.2 +/- 30.9 to 884.5 +/- 210.2 pg/mL (P < .05) at 1 hour and peaked at 3195.3 +/- 476 pg/mL (P < .001) at 4 hours. Before the surge of circulating catecholamines and despite complete beta adrenergic blockade, rhTNF-alpha induced a 7% to 40% increase in LV contractile performance during the 60-minute infusion. After this initial positive inotropic effect, rhTNF-alpha treatment led to precipitous systolic dysfunction between 2 and 7 hours of exposure; this myocardial depressant effect persisted at 25 hours. LV systolic performance declined to 19% to 35% of baseline values, depending on the specific contractile parameter evaluated. We conclude that rhTNF-alpha affects LV systolic function in a time-dependent biphasic manner. Increases in circulating catecholamines after rhTNF-alpha infusion cannot account for the early improvement in LV systolic performance.

CAROTID ATHEROSCLEROSIS PREDICTS INCIDENT ACUTE CORONARY SYNDROMES IN RHEUMATOID ARTHRITIS

OBJECTIVE The role of atherosclerosis in the acute coronary syndromes (ACS) that occur in patients with rheumatoid arthritis (RA) has not been quantified in detail. We undertook this study to determine the extent to which ACS are associated with carotid atherosclerosis in RA. METHODS We prospectively ascertained ACS, defined as myocardial infarction, unstable angina, cardiac arrest, or death due to ischemic heart disease, in an RA cohort. We measured carotid atherosclerosis using high-resolution ultrasound. We used Cox proportional hazards models to estimate the association between ACS and atherosclerosis, adjusting for demographic features, cardiovascular (CV) risk factors, and RA manifestations. RESULTS We performed carotid ultrasound on 636 patients whom we followed up for 3,402 person-years. During this time, 84 patients experienced 121 new or recurrent ACS events, a rate of 3.5 ACS events per 100 patient-years (95% confidence interval [95% CI] 3.0-4.3). Among the 599 patients without a history of ACS, 66 incident ACS events occurred over 3,085 person-years, an incidence of 2.1 ACS events per 100 person-years (95% CI 1.7-2.7). The incidence of new ACS events per 100 patient-years was 1.1 (95% CI 0.6-1.7) among patients without plaque, 2.5 (95% CI 1.7-3.8) among patients with unilateral plaque, and 4.3 (95% CI 2.9-6.3) among patients with bilateral plaque. Covariates associated with incident ACS events independent of atherosclerosis included male sex, diabetes mellitus, and a cumulative glucocorticoid dose of ≥ 20 gm. CONCLUSION Atherosclerosis is strongly associated with ACS in RA. RA patients with carotid plaque, multiple CV risk factors (particularly diabetes mellitus or hypertension), many swollen joints, and a high cumulative dose of glucocorticoids, as well as RA patients who are men, are at high risk of ACS.

Regulation of CCAAT/Enhancer Binding Protein, Interleukin-6, Interleukin-6 Receptor, and gp130 Expression During Myocardial Ischemia/Reperfusion

BACKGROUND Interleukin (IL)-6 is elevated in myocardium after ischemia and reperfusion. The IL-6 promoter/enhancer region contains response elements for nuclear factor-kappaB, AP-1, and CCAAT/enhancer binding protein (C/EBP). Expression and regulation of C/EBP in rat myocardium after ischemia and reperfusion has not been defined, nor has the behavior of the specific IL-6 receptor (IL-6R) or the signal transducer gp130. METHODS AND RESULTS C/EBP DNA binding activity was not detected in shams or in previously ischemic tissue at 15 minutes of reperfusion; it was detected at 30 minutes of reperfusion, increased at 1 hour of reperfusion, and declined by 6 hours of reperfusion. A supershift was observed with anti-C/EBP-beta but not with anti-alpha or anti-delta antibodies. mRNA and protein levels of IL-6 and gp130 were detected at low levels in controls, increased at 1 hour of reperfusion, and remained high until 6 hours of reperfusion. IL-6R mRNA and protein were not detected in controls, but its mRNA was induced at 1 hour of reperfusion and its protein at 2 hours of reperfusion. Although effects of reperfusion were rapid, in ischemic tissue not reperfused, low levels of C/EBP were detected at 4 hours, and at 24 hours the levels were slightly elevated. Significant upregulation in IL-6, IL-6R, and gp130 occurred only at 24 hours of sustained ischemia. CONCLUSIONS Reperfusion after a brief period of ischemia caused induction of myocardial C/EBP (beta-subunit). The rapid and sustained production of IL-6 with concomitant expression of IL-6 receptor and gp130 suggest that these factors may participate in a local inflammatory cascade after myocardial ischemia and reperfusion.

Inhibition of nuclear factor κB attenuates proinflammatory cytokine and inducible nitric-oxide synthase expression in postischemic myocardium

We have previously reported that induction of nuclear factor-kappa B (NF-kappa B) occurs in a biphasic manner in postischemic myocardium. Because interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-alpha), and inducible nitric-oxide synthase (iNOS) contain kappa B-response elements, and since transforming growth factor-beta 1 (TGF-beta 1) down-modulates both cytokine and iNOS expression, we studied their temporal expression during myocardial ischemia/reperfusion (I/R). Northern and Western analyses showed low levels of IL-6 and no signal for IL-1 beta, TNF-alpha and iNOS under basal conditions. Their expression rose significantly over sham-operated controls by 1 h reperfusion, and persisted high for various periods. Under basal conditions, low levels of TGF-beta 1 were detected, which rose significantly at 3 h reperfusion, and remained high until 24 h reperfusion. Administration of diethyldithiocarbamate (DDC) inhibited induction of NF-kappa B and concomitantly the expression of IL-1 beta, IL-6, TNF-alpha as well as iNOS. However, expression of TGF-beta was not altered. Our results indicate that ischemia/reperfusion induces NF-kappa B, and upregulates kappa B-response genes. Administration of DDC inhibits NF-kappa B levels, and attenuates expression of inflammatory cytokines and iNOS.

TNF-α and H2O2 induce IL-18 and IL-18Rβ expression in cardiomyocytes via NF-κB activation

Abstract Myocardial ischemia/reperfusion is characterized by oxidative stress and induction of proinflammatory cytokines. Interleukin (IL)-18, a member of the IL-1 family, acts as a proinflammatory cytokine, and is induced during various immune and inflammatory disorders. Therefore, in the present study we investigated whether IL-18 expression is regulated by cytokines and oxidative stress in cardiomyocytes. TNF-α induced rapid and sustained activation of NF-κB whereas H 2 O 2 induced delayed and transient activation. Both TNF-α and H 2 O 2 induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-α led to sustained expression. Expression of IL-18Rβ, but not α, was induced by both agonists. TNF-α and H 2 O 2 induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-α and H 2 O 2 induced IL-18 and IL-18Rβ, but not basal expression of IL-18Rα. These results indicate that adult cardiomyocytes express IL-18 and its receptors, and proinflammatory cytokines and oxidative stress regulate their expression via activation of NF-κB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.

H2O2 activates Nox4 through PLA2-dependent arachidonic acid production in adult cardiac fibroblasts

Stimulated production of reactive oxygen species (ROS) by plasma membrane‐associated nicotinamide adenine dinucleotide phosphate oxidases (Nox) in non‐phagocytic cells regulates a number of biological processes including growth, vessel tone, and oxygen sensing. The purpose of this study was to investigate H2O2‐stimulated ROS production in primary adult cardiac fibroblasts (CF). Results demonstrate that CF express an H2O2‐inducible oxidant generating system that is inhibitable by diphenylene iodonium (DPI) and sensitive to antioxidants. In addition to H2O2, generation of ROS was stimulated potently by 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and arachidonic acid (AA) in a protein kinase C‐independent manner. Pretreatment with arachidonyl trifluoromethyl ketone was nearly as effective as DPI at reducing H2O2‐ and OAG‐stimulated oxidant generation indicating a central role for phospholipase A2 (PLA2) in this signaling pathway. Co‐stimulation with H2O2 and OAG did not increase ROS generation as compared to OAG alone suggesting both agonists signal through a shared, rate‐limited enzymatic pathway involving PLA2. Co‐stimulation with H2O2 and AA had additive effects indicating these two agonists stimulate oxidant production through a parallel activation pathway. Reverse transcriptase‐coupled polymerase chain reaction and Western blotting demonstrate primary cardiac fibroblasts express transcripts and protein for Nox4, p22, p47, and p67 phox. Transfections with Nox4 small inhibitory ribonucleic acid oligonucleotides or p22 phox antisense oligonucleotides significantly downregulated stimulated Nox activity. Inhibitors of nitric oxide synthases were without effect. We conclude adult CF express Nox4/p22 phox‐containing oxidant generating complex activated by H2O2, OAG, and AA through a pathway that requires activation of PLA2.