Resident Cardiac Macrophages Research Articles

Immune-Electrical Connection

Macrophages interact with cardiomyocytes in the AV node via Cx43 gap junctions and assist normal AV nodal conduction, and macrophage depletion promotes AV block. Macrophages interact with cardiomyocytes in the AV node via Cx43 gap junctions and assist normal AV nodal conduction, and macrophage depletion promotes AV block. CITATION Hulsmans M, Clauss S, Xiao L, et al. Macrophages facilitate electrical conduction in the heart. Cell 2017; 169: 510–522. While the phagocytic role of macrophages in fighting pathogens and foreign environmental antigens is well known, the function of tissue-resident macrophages is less well understood. Cardiac-resident macrophages are yolk sac–derived rather than circulating monocyte–derived, and they self-renew via proliferation of local progenitors. They have been shown to play a role in tissue repair following heart tissue injury, but it is not clear whether they have a function at steady state. Using elegant genetic mouse models, single-cell RNA sequencing (RNA-Seq) and mathematical modeling, Hulsmans and colleagues demonstrate that cardiac-resident macrophages form gap junctions with cardiomyocytes in the atrioventricular (AV) node and help electrical conduction independent of the autonomic nervous system. The authors first demonstrated that the distal AV node, but not the left ventricle, is rich in macrophages, both in mice (CD11b+ F4/80+ Ly6Clo CD64+ CX3CR1+cells) and in humans (CD68+ CD163+ cells). Use of Cx3cr1GFP reporter mice enabled the investigators to visualize the cells’ elongated form in close contact with cardiomyocytes, whereas single-cell RNA-Seq demonstrated their enrichment in genes associated with conduction. Immunofluorescence and electron microscopy revealed an average of three punctate gap junctions/interactions between macrophages and cardiomyocytes, composed of the connexin 43 (Cx43) protein. Using whole-cell patch clamp, the authors showed that cardiomyocytes were depolarized when cultured in the presence of cardiac macrophages compared with cultured alone, whereas a fraction of the cocultured macrophages showed rhythmic depolarization and had a more negative resting membrane potential compatible with electrical coupling. Pharmacological blockade of Cx43 reversed the depolarization of cardiomyocytes cocultured with macrophages. Mathematical modeling recapitulated the observed more positive resting membrane potential of cardiomyocytes when associated with macrophages, which should facilitate myocyte depolarization with less stimulation; moreover, increasing coupling of macrophages/cardiomyocytes predicted accelerated cardiomyocyte repolarization, which should result in a shorter refractory period. Together, these alterations should assist AV conduction at higher frequencies. Indeed, forced depolarization of macrophages via photogenetics (in hearts from mice with tamoxifen-induced expression in macrophages of the photoactivatable cation channelrhodopsin 2, such that illuminating the AV node resulted in a Na+ influx) improved AV node conduction during rapid pacing, as measured by electrocardiogram. Finally, the authors used complementary mouse models to address the necessity of macrophage/cardiomyocyte electrical coupling for normal AV nodal conduction. Depletion of Cx43 in macrophages (Cx3cr1 Cx43−/−) promoted first- and second-degree AV block, as did use of CSF1op mice lacking tissue macrophages in many organs. Importantly for mouse immunologists who often use this genetic approach, diphtheria-induced depletion of macrophages in Cd11bDTR mice resulted in first- to third-degree AV block starting one day after diphtheria toxin (DT) injection, in a DT dose–dependent manner. In contrast, macrophage ablation following clodronate liposome injection had no detectable effect, presumably due to the very partial deletion effected by this approach. Thus, overall, macrophages in the distal AV node associate with cardiomyocytes via Cx43 gap junctions and are required for normal AV electrical conduction (Figure 1). These unanticipated results have implications for cardiac transplantation. Ischemia/reperfusion injury and inflammation are likely to change the phenotype and function of resident macrophages and thus, perhaps, alter their contribution to electrical conduction, which might result in threatening arrhythmias. Indeed, IL-1β has been associated with arrhythmias in diabetes-associated macrophage activation. More research needs to be devoted to the outcome of donor resident macrophages after transplantation. Do they continue to self-renew from local precursors, or are they replaced by host macrophages and, if so, can they establish gap junctions with donor cardiomyocytes? Certainly, arrhythmias and AV block are not uncommon after cardiac transplantation. Mouse experiments suggest that, after transplantation, donor class II disappears over time and T cells with direct reactivity to donor class II cease to be activated after a few weeks. This may suggest that donor resident macrophages are eliminated and replaced by host cells or that their expression of class II quiesces sufficiently to avoid direct T cell activation. A closer look at whether donor and/or host macrophages connect with donor myocytes at different times after transplantation and how they influence AV conduction may help optimize the monitoring of heart transplant recipients.

Cardiac resident macrophages are involved in hypoxia‑induced postnatal cardiomyocyte proliferation.

Induction of cardiomyocyte proliferation, the most promising approach to reverse myocardial attrition, has been gaining importance as a therapy for cardiovascular disease. Hypoxia and macrophages were previously independently reported to promote cardiomyocyte proliferation in mice. However, whether hypoxia promotes cardiomyocyte proliferation in humans, and the association between hypoxia and macrophages in cardiomyocyte proliferation, have not to the best of our knowledge been previously investigated. The present study investigated the cardiomyocyte proliferation in 22 acyanotic and 29 cyanotic patients. Cardiomyocyte proliferation in a hypoxic mouse model (15% O2) was subsequently performed and the macrophage subsets were analyzed. A C-C chemokine receptor type 2 (CCR2) inhibitor was used to increase the number of resident macrophages in order to investigate the effect of macrophages on cardiomyocyte proliferation. The results demonstrated that cardiomyocyte proliferation in the cyanotic infant group was significantly increased compared with the acyanotic infant group and the hypoxia-treated C57BL/6J neonates confirmed the hypoxia-induced cardiomyocyte proliferation. However, hypoxia did not induce the proliferation of isolated cardiomyocytes. Notably, hypoxia treatment increased the number of cardiac resident macrophages in neonate hearts. Furthermore, increasing the number of resident macrophages significantly enhanced cardiomyocyte proliferation. In conclusion, postnatal hypoxia promoted cardiomyocyte proliferation in humans and animals, and cardiac resident macrophages may be involved in this process. Therefore, this novel mechanism may provide a promising strategy for cardiovascular disease treatment.

A novel 72-kDa leukocyte-derived osteoglycin enhances the activation of toll-like receptor 4 and exacerbates cardiac inflammation during viral myocarditis

BackgroundViral myocarditis can severely damage the myocardium through excessive infiltration of immune cells. Osteoglycin (OGN) is part of the small leucine-rich repeat proteoglycan (SLRP) family. SLRP’s may affect inflammatory and fibrotic processes, but the implication of OGN in cardiac inflammation and the resulting injury upon viral myocarditis is unknown.Methods and resultsThis study uncovered a previously unidentified 72-kDa variant of OGN that is predominant in cardiac human and mouse samples of viral myocarditis. Its absence in mice significantly decreased cardiac inflammation and injury in Coxsackievirus-B3-induced myocarditis. It also delayed mortality in lipopolysaccharide-induced endotoxemia going along with a reduced systemic production of pro-inflammatory cytokines. This 72-kDa OGN is expressed in the cell membrane of circulating and resident cardiac macrophages and neutrophils. Co-immunoprecipitation and OGN siRNA experiments revealed that this 72-kDa variant activates the toll-like receptor-4 (TLR4) with a concomitant increase in IL-6, TNF-α, IL-1β, and IL-12 expression. This immune cell activation by OGN occurred via MyD88 and increased phosphorylation of c-jun. Finally, the 72-kDa chondroitin sulfate is the result of O-linked glycosylation of the 32-kDa protein core of OGN. In contrast, the 34-kDa dermatan sulfate-OGN, involved in collagen cross linking, was also the result of O-linked glycosylation.ConclusionThe current study discovered a novel 72-kDa chondroitin sulfate-OGN that is specific for innate immune cells. This variant is able to bind and activate TLR4. The absence of OGN decreases cytokine production by both circulating and cardiac leukocytes upon (systemic) LPS exposure, and reduces cardiac inflammation and injury in viral myocarditis.

Abstract 467: Glucose Promotes M2-related Transcriptional Reprogramming of Resident Cardiac Macrophages in Response to Hypothermic Ischemic Arrest and Reperfusion

Stress hyperglycemia and inflammation frequently develop in open heart surgery patients. Although both factors independently contribute to increased peri-operative morbidity and mortality, the impact of high glucose levels on cardiac inflammatory response remains unknown. We investigated the isolated working rat heart as a model to study cardiac early stress response to surgery. Hearts of male Sprague Dawley rats were cold-arrested and subjected to 60 minutes normothermic reperfusion in the working mode with Krebs-Henseleit buffer supplemented with ketone bodies and propionate plus glucose (25 mM) or mannitol (25 mM; osmotic control). Alterations of gene expression in the left ventricle were determined by microarray and real-time PCR analyses. Compared to non-perfused hearts, perfused hearts displayed a more than twofold increased expression for 71 genes (mannitol group) and 103 genes (glucose group) connected to inflammation, cell proliferation, and apoptosis. The transcriptional changes were highly similar to gene alterations previously reported in the right atrium ( P < 2.34E-16) and left ventricle ( P < 4.83E-46) of patients who underwent cardiac surgery with cardiopulmonary bypass. Pathway analysis with Reactome revealed an up-regulation of metabolic processes associated with the proliferation and activation of immune cells, including glycolysis, glutaminolysis, fatty acid synthesis, polyamine synthesis, and hexosamine synthesis. Although the transcriptional remodeling occurred independently from the presence of glucose, glucose significantly increased further the expression of several transcription factors and markers associated with M2 polarization of macrophages, including Myc (1.6-fold), Nr4a1 (1.3-fold), Nr4a2 (1.8-fold), Zc3h12a (1.3-fold), Fosl2 (1.4-fold), Cebpb (1.2-fold), and Arg1 (1.7-fold). Interestingly, glucose failed to enhance the expression of M2-related genes in the heart of rats rendered insulin resistant by high-sucrose feeding. Besides demonstrating that the isolated working rat heart accurately reproduces the stress response associated with open heart surgery, the results also suggest that glucose promotes the alternative activation of resident cardiac macrophages in the stressed heart.

Implications for the role of macrophages in a model of myocardial fibrosis: CCR2(-/-) mice exhibit an M2 phenotypic shift in resident cardiac macrophages.

Macrophages (MΦ) are functionally diverse and dynamic. Until recently, cardiac MΦ were assumed to be monocyte derived; however, resident cardiac MΦ (rCMΦ), present at baseline, were identified in myocardia and have been implicated in cardiac healing. Previously, we demonstrated that CCR2(-/-) mice are protected from myocardial fibrosis - an observation initially attributed to changes in infiltrating monocytes. Here, we reexplored this observation in the context of our new understanding of rCMΦ. Male CCR2(-/-) and C57BL/6 hearts were digested and purified to a single cell suspension, incubated with fluorophore-linked antibodies (CCR2, CX3CR1, CD11b, Ly6C, TNF-α, and IL-10), and assessed by flow cytometry. Differentiated MΦ were cocultured with fibroblasts in order to characterize how MΦ phenotype influences fibroblast activation. Fibroblasts were characterized for their expression of smooth muscle cell actin (SMA). A significant decrease in Ly6C expression was observed in the CCR2(-/-) cardiac MΦ population relative to WT, which corresponded with significantly lower TNF-α expression and significantly higher IL-10 expression. Using in vitro coculture system, classical MΦ promoted fibroblast activation relative to nonclassical MΦ. CCR2(-/-) rCMΦ favor a more antiinflammatory phenotype relative to WT controls. Moreover, a shift toward the antiinflammatory promotes proliferation, but not activation in vitro. Together, these observations suggest that antiinflammatory cardiac MΦ populations may inhibit myocardial fibrosis in a pathological setting by preventing the activation of fibroblasts. Here, we provide novel evidence for baseline differences in rCMΦ phenotypes (i.e. classical vs. nonclassical) and how these differences could modulate cardiac healing. Importantly, we observed differences in how classical vs. nonclassical MΦ influenced fibroblast activation, which could, in turn, affect fibrosis.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Abstract 55: Opposing Roles Of Distinct Macrophage Lineages In Heart Failure And Cardiac Recovery

Why inflammation is simultaneously deleterious following injury and essential for tissue repair continues to be fundamentally important and debated question. Recently, a new paradigm has emerged in the macrophage field: that organs are replete with resident macrophages of embryonic origin, distinct from monocyte-derived macrophages. This added complexity raises the question of whether distinct immune cells drive inflammatory and reparative activities following injury. Previous work has demonstrated that the neonatal heart has a remarkable capacity for tissue repair compared to the adult, offering an ideal context to examine these concepts. We hypothesized that unrecognized differences in macrophage composition in the neonatal and adult heart represents a key determinant of cardiac recovery. To test this hypothesis, we generated a novel cardiomyocyte ablation model and demonstrated that following injury neonatal mice expand a population of resident cardiac macrophages derived from embryonic lineages, which generate minimal inflammation and are necessary and sufficient for cardiac recovery through promotion of cardiomyocyte proliferation and angiogenesis. During homeostasis the adult heart also contained embryonic-derived macrophages with similar properties. However, following injury these cells disappeared, and instead, the adult heart recruited pro-inflammatory monocytes and monocyte-derived macrophages that lacked reparative activities. Inhibition of monocyte recruitment into the injured adult heart preserved embryonic-derived macrophage subsets, reduced inflammatory cytokine and chemokine production, and enhanced tissue repair. Together, these findings indicate that embryonic-derived macrophages, rather than monocyte-derived macrophages, are key mediators of cardiac recovery. Therapeutics targeting distinct macrophage and monocyte lineages may serve as novel treatments for heart failure.

Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.

Macrophages populate the steady-state myocardium. Previously, all macrophages were thought to arise from monocytes; however, it emerged that, in several organs, tissue-resident macrophages may self-maintain through local proliferation. Our aim was to study the contribution of monocytes to cardiac-resident macrophages in steady state, after macrophage depletion in CD11b(DTR/+) mice and in myocardial infarction. Using in vivo fate mapping and flow cytometry, we estimated that during steady state the heart macrophage population turns over in ≈1 month. To explore the source of cardiac-resident macrophages, we joined the circulation of mice using parabiosis. After 6 weeks, we observed blood monocyte chimerism of 35.3±3.4%, whereas heart macrophages showed a much lower chimerism of 2.7±0.5% (P<0.01). Macrophages self-renewed locally through proliferation: 2.1±0.3% incorporated bromodeoxyuridine 2 hours after a single injection, and 13.7±1.4% heart macrophages stained positive for the cell cycle marker Ki-67. The cells likely participate in defense against infection, because we found them to ingest fluorescently labeled bacteria. In ischemic myocardium, we observed that tissue-resident macrophages died locally, whereas some also migrated to hematopoietic organs. If the steady state was perturbed by coronary ligation or diphtheria toxin-induced macrophage depletion in CD11b(DTR/+) mice, blood monocytes replenished heart macrophages. However, in the chronic phase after myocardial infarction, macrophages residing in the infarct were again independent from the blood monocyte pool, returning to the steady-state situation. In this study, we show differential contribution of monocytes to heart macrophages during steady state, after macrophage depletion or in the acute and chronic phase after myocardial infarction. We found that macrophages participate in the immunosurveillance of myocardial tissue. These data correspond with previous studies on tissue-resident macrophages and raise important questions on the fate and function of macrophages during the development of heart failure.

Cardioprotective function of cardiac macrophages

The heart is composed of several cell types including cardiomyocytes, cardiac fibroblasts, endothelial, and smooth muscle cells. In addition to these major cell types, cardiac macrophages are also present in small numbers under physiological conditions. Recently, the resident macrophage is considered to have vital functions in the maintenance of tissues and homeostasis in many organs, including brain, liver, adipose tissue, lymphatic tissue, and intestinal tract. However, detailed functions of the cardiac resident macrophage are not fully understood. Although the removal of debris arising from damaged cardiomyocytes and pro-inflammatory effects after heart injuries are conventional tasks of cardiac macrophages (classically activated macrophage or M1 macrophage), novel functions like anti-inflammatory roles, adaptive response, and tissue maintenance have also been reported in recent years. Macrophages that possess these novel functions are generally so-called M2 macrophages, which are alternatively activated and show anti-inflammatory phenotype under pathological conditions. In this review, we focus on the cardioprotective function of the cardiac macrophage and discuss in light of unveiled fundamental functions of macrophages that have been also found in other organs.

Embryonic and Adult-Derived Resident Cardiac Macrophages Are Maintained through Distinct Mechanisms at Steady State and during Inflammation

Cardiac macrophages are crucial for tissue repair after cardiac injury but are not well characterized. Here we identify four populations of cardiac macrophages. At steady state, resident macrophages were primarily maintained through local proliferation. However, after macrophage depletion or during cardiac inflammation, Ly6c(hi) monocytes contributed to all four macrophage populations, whereas resident macrophages also expanded numerically through proliferation. Genetic fate mapping revealed that yolk-sac and fetal monocyte progenitors gave rise to the majority of cardiac macrophages, and the heart was among a minority of organs in which substantial numbers of yolk-sac macrophages persisted in adulthood. CCR2 expression and dependence distinguished cardiac macrophages of adult monocyte versus embryonic origin. Transcriptional and functional data revealed that monocyte-derived macrophages coordinate cardiac inflammation, while playing redundant but lesser roles in antigen sampling and efferocytosis. These data highlight the presence of multiple cardiac macrophage subsets, with different functions, origins, and strategies to regulate compartment size.

Free heme is a danger signal inducing expression of proinflammatory proteins in cultured cells derived from normal rat hearts

Endogenous molecules from damaged tissue act as danger signals to trigger or amplify the immune/inflammatory response. In this study, we examined whether free heme induced pro-inflammatory proteins in cultured cells derived from normal hearts and investigated the cells targeted by heme, together with its mechanism of action in these cells. We cultured collagenase-isolated heart-derived cells from normal rats and examined whether free heme induced pro-inflammatory proteins, reactive oxygen species (ROS) production and NF-κB activation, by quantitative RT-PCR, ELISA and flow cytometry. Free heme increased mRNA of various pro-inflammatory proteins in cultured cardiac resident cells (CCRC) (at least 100-fold) and induced intracellular ROS formation. Approximately 85–90% of CCRC are fibroblast/smooth muscle cells and 10–15% are CD11bc-positive macrophages; therefore to examine individual target cells, macrophage-deleted (CD11bc-negative) CCRC, primary cultured cells (cardiac fibroblasts, arterial smooth muscle cells and cardiac microvascular endothelial cells) and macrophage cells lines (NR8383) were similarly treated. Free heme activated NF-κB and induced expression of some pro-inflammatory proteins, including IL-1 and TNF-α in NR8383. On the other hand, macrophage-deleted CCRC strongly increased expression of these proteins on treatment with IL-1 or TNF-α, but not free heme. Induction of expression of pro-inflammatory proteins by free heme was not inhibited by intracellular ROS reduction, but by protease and proteasome inhibitors capable of regulating NF-κB. These data suggest that free heme strongly induces various pro-inflammatory proteins in injured hearts through NF-κB activation in cardiac resident macrophages and through cross-talk between macrophages and fibroblast/smooth muscle cells mediated inter alia by IL-1, TNF-α.

Tumor necrosis factor-a, biologic agents and cardiovascular risk

The increased risk of premature cardiovascular disease (CVD) in rheumatoid arthritis (RA) patients may depend on traditional risk factors but may also be attributable to RA-specific risk factors such as disease-related dyslipidemia, or cytokines such as tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is a proinflammatory cytokine that can produce widespread deleterious effects when expressed in large amounts. It is produced in the heart by both cardiac myocytes and resident macrophages under conditions of cardiac stress, and is thought to be responsible for many of the untoward manifestations of cardiac disease. TNF-alpha may play a role in the triggering and perpetuation of atherosclerosis. Treatment with biologic agents directed against TNF-alpha has significant clinical benefits in inflammatory diseases such as RA and may be able to reduce cardiovascular risk. The disappointing results of the recent studies to antagonize TNF-alpha in CVD may have various explanations. However, the effects of TNF-alpha blockers on incident cases of congestive heart failure (CHF) in RA remains controversial. Due to the lack of evidence of a beneficial effect of anti-TNF-alpha agents in treatment of CHF, they should not be used to treat patients with New York Heart Association (NYHA) class III or IV heart failure.

The role of tumor necrosis factor in cardiac disease.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that can produce widespread deleterious effects when expressed in large amounts. It is produced in the heart by both cardiac myocytes and resident macrophages under conditions of cardiac stress, and is thought to be responsible for many of the untoward manifestations of cardiac disease. This article discusses the role of TNF in heart disease and some potential therapeutic modalities that can influence the cytokine activity. The results of controlled studies would suggest that TNF inhibition does not influence the clinical course of patients with heart failure.

Inhibition of myocardial TNF-alpha production by heat shock. A potential mechanism of stress-induced cardioprotection against postischemic dysfunction.

Overproduction of tumor necrosis factor-alpha (TNF-alpha) contributes to cardiac dysfunction associated with systemic or myocardial stress, such as endotoxemia and myocardial ischemia/reperfusion (I/R). Heat shock has been demonstrated to enhance cardiac functional resistance to I/R. However, the protective mechanisms remain unclear. The purpose of this study was to determine: (1) whether cardiac macrophages express heat shock protein 72 (HSP72) after heat shock, (2) whether induced cardiac HSP72 suppresses myocardial TNF-alpha production during I/R, and (3) whether preservation of postischemic myocardial function by heat shock is correlated with attenuated TNF-alpha production during I/R. Rats were subjected to heat shock (42 degrees C for 15 min) and 24 h recovery. Immunoblotting confirmed the expression of cardiac HSP72. Immunofluorescent staining detected HSP72 in cardiac interstitial cells including resident macrophages rather than myocytes. Global I/R caused a significant increase in myocardial TNF-alpha. The increase in myocardial TNF-alpha was blunted by prior heat shock and the reduced myocardial TNF-alpha level was correlated with improved cardiac functional recovery. This study demonstrates for the first time that heat shock induces HSP72 in cardiac resident macrophages and inhibits myocardial TNF-alpha production during I/R. These observations suggest that inhibition of myocardial TNF-alpha production may be a mechanism by which HSP72 protects the heart against postischemic dysfunction.

Histochemical and morphological characteristics of canine cardiac mast cells.

Cardiac mast cells have been recently isolated and characterized in humans, however canine cardiac mast cells have not been investigated. The objective of this study is to describe the histological and morphological characteristics of canine cardiac mast cells and examine the potential usefulness of canine models in investigating the role of mast cells in cardiovascular pathology. Canine cardiac mast cells could be easily identified by staining with Toluidine Blue or FITC-avidin. Using Toluidine Blue staining, we demonstrated fewer mast cells in formalin-fixed samples than in specimens fixed in Carnoy's, thus identifying a formalin-sensitive mast cell population in the canine heart. Mast cells were equally distributed in atria and ventricles with approximately 50% showing a perivascular location. Using enzyme-histochemical techniques, we detected tryptase and chymase activity in canine cardiac mast cells. Ultrastructural studies identified mast cells as granular cells with an eccentric non-segmented nucleus. Immunohistochemistry with the macrophage specific antibody AM-3K demonstrated that resident cardiac macrophages were 1.9 times more numerous than mast cells, also showing a predominantly perivascular (60%) location. Perivascular macrophages were more often periarteriolar, whereas perivascular mast cells were more often located along small veins and capillaries. Due to their ability to release cytokines and growth factors and their strategic perivascular location, resident cardiac inflammatory cells, such as mast cells and macrophages, may be important in pathological processes causing myocardial inflammation and fibrosis. Furthermore, mast cell-derived chymase, an important angiotensin II-forming enzyme may have a significant role in regulating the cardiac renin-angiotensin system.

Adenosine Reduces Cardiac TNF-α Production and Human Myocardial Injury Following Ischemia-Reperfusion

Myocardial tumor necrosis factor-α (TNF-α) is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion injury (I/R), sepsis, chronic heart failure, and cardiac allograft rejection. Cardiac resident macrophages and cardiomyocytes themselves produce TNF-α. In this regard, adenosine (ADO) has been reported to reduce macrophage TNF-α production. Our purposes were to determine whether (1) I/R induces rat myocardial TNF-α production; (2) ADO decreases ischemia-induced rat myocardial TNF-α production; (3) ADO functionally protects human myocardium against I/R; and (4) TNF-α-binding protein (TNFBP; p55) confers similar protection when substituted for ADO pretreatment. To study this, human atrial trabeculae were obtained during cardiac surgery and suspended in organ baths, paced at 1 Hz, and force development was recorded during I/R (45/120 min) with or without ADO pretreatment (125 μM × 10 min), or TNFBP (1 μg/ml) during I/R. Isolated rat hearts were perfused using the Langendorff method undergoing I/R (20/40 min) with or without ADO pretreatment (125 μM × 2 min) and rat myocardial expression of TNF-α was assessed by ELISA. Results demonstrated that I/R increased rat myocardial TNF-α levels from 324 ± 36 to 902 ± 77 pg/g (P< 0.05; ANOVA and Bonferroni/Dunn) and decreased human myocardial developed force (DF) to 18 ± 2% of baseline (%BDF;P< 0.05). ADO pretreatment decreased ischemia-induced rat myocardial TNF-α production (356 ± 107 pg/g;P< 0.05) and increased postischemic DF of human myocardium to 39 ± 3% BDF (P< 0.05). Further substantiating the link between ischemia-induced TNF-α production and injury, TNFBP administration similarly improved post-I/R function of human myocardium (55 ± 5% BDF;P< 0.05 vs. I/R alone). We conclude that (1) I/R induces rat myocardial TNF-α production; (2) ADO pretreatment decreases I/R-induced rat myocardial TNF-α production; (3) ADO improves human myocardial function; (4) TNFBP confers similar protection; and (5) inhibition/ neutralization of TNF-α represents a novel strategy for protecting human myocardium against ischemia and reperfusion injury.

Adenosine decreases post-ischaemic cardiac TNF-alpha production: anti-inflammatory implications for preconditioning and transplantation.

Tumour necrosis factor-alpha (TNF-alpha) is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischaemia-reperfusion injury (I/R), sepsis, chronic heart failure and cardiac allograft rejection. Cardiac resident macrophages, infiltrating leucocytes, and cardiomyocytes themselves produce TNF-alpha. Although adenosine reduces macrophage TNF-alpha production and protects myocardium against I/R, it remains unknown whether I/R induces an increase in cardiac TNF-alpha in a crystalloid-perfused model (in the absence of blood), and, whether adenosine decreases cardiac TNF-alpha and protects function after I/R. To study this, isolated rat hearts were crystalloid-perfused using the Langendorff method and subjected to I/R, with or without adenosine pretreatment. Post-ischaemic cardiac TNF-alpha (enzyme-linked immunosorbent assay and bioassay) and function were determined (Langendorff). I/R increased cardiac TNF-alpha and impaired myocardial function. Adenosine decreased cardiac TNF-alpha and improved post-ischaemic functional recovery. This study demonstrates that: first, I/R induces an increase in cardiac tissue TNF-alpha in a crystalloid-perfused model: second, adenosine decreases cardiac TNF-alpha and improves post-ischaemic myocardial function; third, decreased cardiac TNF-alpha may represent a mechanism by which adenosine protects myocardium; and fourth, adenosine-induced suppression of cardiac TNF-alpha may provide an anti-inflammatory link to preconditioning and have implications for cardiac allograft preservation.