Delayed expression of cytokines after reperfused myocardial infarction: possible trigger for cardiac dysfunction and ventricular remodeling

Previous studies have shown that 1 wk after permanent coronary artery ligation in rats, some cellular mechanisms involving TNF-alpha occur and contribute to the development of cardiac dysfunction and subsequent heart failure. The aim of the present study was to determine whether similar phenomena also occur after ischemia-reperfusion and whether cytokines other than TNF-alpha can also be involved. Anesthetized male Wistar rats were subjected to 1 h coronary occlusion followed by reperfusion. Cardiac geometry and function were assessed by echocardiography at days 5, 7, 8, and 10 postligation. Before death, heart function was assessed in vivo under basal conditions, as well as after volume overload. Finally, hearts were frozen for histoenzymologic assessment of infarct size and remodeling. The profile of cardiac cytokines was determined by ELISA and ChemiArray on heart tissue extracts. As expected, ischemia-reperfusion induced a progressive remodeling of the heart, characterized by left ventricular free-wall thinning and cavity dilation. Heart function was also decreased in ischemic rats during the first week after surgery. Interestingly, a transient and marked increase in TNF-alpha, IL-1beta, IL-6, cytokine-induced neutrophil chemoattractant (CINC) 2, CINC3, and macrophage inflammatory protein-3alpha was also observed in the myocardium of myocardial ischemia (MI) animals at day 8, whereas the expression of anti-inflammatory interleukins IL-4 and IL-10 remained unchanged. These results suggest that overexpression of proinflammatory cytokines occurring during the first week after ischemia-reperfusion may play a role in the adaptative process in the myocardium and contribute to early dysfunction and remodeling.     

IL-10 is induced in the reperfused myocardium and may modulate the reaction to injury

Reperfusion of the ischemic myocardium is associated with a dramatic inflammatory response leading to TNF-alpha release, IL-6 induction, and subsequent neutrophil-mediated cytotoxic injury. Because inflammation is also an important factor in cardiac repair, we hypothesized the presence of components of the inflammatory reaction with a possible role in suppressing acute injury. Thus, we investigated the role of IL-10, an anti-inflammatory cytokine capable of modulating extracellular matrix biosynthesis, following an experimental canine myocardial infarction. Using our canine model of myocardial ischemia and reperfusion, we demonstrated significant up-regulation of IL-10 mRNA and protein in the ischemic and reperfused myocardium. IL-10 expression was first detected at 5 h and peaked following 96-120 h of reperfusion. In contrast, IL-4 and IL-13, also associated with suppression of acute inflammation and macrophage deactivation, were not expressed. In the ischemic canine heart, CD5-positive lymphocytes were the predominant source of IL-10 in the myocardial infarct. In the absence of reperfusion, no significant induction of IL-10 mRNA was noted. In addition, IL-12, a Th1-related cytokine associated with macrophage activation, was not detected in the ischemic myocardium. In vitro experiments demonstrated late postischemic cardiac-lymph-induced tissue inhibitor of metalloproteinases (TIMP)-1 mRNA expression in isolated canine mononuclear cells. This effect was inhibited when the incubation contained a neutralizing Ab to IL-10. Our findings suggest that lymphocytes infiltrating the ischemic and reperfused myocardium express IL-10 and may have a significant role in healing by modulating mononuclear cell phenotype and inducing TIMP-1 expression.     

Systematic Characterization of Myocardial Inflammation,Repair, and Remodeling in a Mouse Model of Reperfused Myocardial Infarction

Mouse models of myocardial infarction are essential tools for the study of cardiac injury, repair, and remodeling. Our current investigation establishes a systematic approach for quantitative evaluation of the inflammatory and reparative response, cardiac function, and geometry in a mouse model of reperfused myocardial infarction. Reperfused mouse infarcts exhibited marked induction of inflammatory cytokines that peaked after 6 hr of reperfusion. In the infarcted heart, scar contraction and chamber dilation continued for at least 28 days after reperfusion; infarct maturation was associated with marked thinning of the scar, accompanied by volume loss and rapid clearance of cellular elements. Echocardiographic measurements of end-diastolic dimensions correlated well with morphometric assessment of dilative remodeling in perfusion-fixed hearts. Hemodynamic monitoring was used to quantitatively assess systolic and diastolic function; the severity of diastolic dysfunction following myocardial infarction correlated with cardiomyocyte hypertrophy and infarct collagen content. Expression of molecular mediators of inflammation and cellular infiltration needs to be investigated during the first 72 hr, whereas assessment of dilative remodeling requires measurement of geometric parameters for at least four weeks after the acute event. Rapid initiation and resolution of the inflammatory response, accelerated scar maturation, and extensive infarct volume loss are important characteristics of infarct healing in mice.     

Inflammatory cytokines associated with cancer growth induce mitochondria and cytoskeleton alterations in cardiomyocytes

Cardiac dysfunction is often observed in patients with cancer also representing a serious problem limiting chemotherapeutic intervention and even patient survival. In view of the recently established role of the immune system in the control of cancer growth, the present work has been undertaken to investigate the effects of a panel of the most important inflammatory cytokines on the integrity and function of mitochondria, as well as of the cytoskeleton, two key elements in the functioning of cardiomyocytes. Either mitochondria features or actomyosin cytoskeleton organization of in vitro-cultured cardiomyocytes treated with different inflammatory cytokines were analyzed. In addition, to investigate the interplay between tumor growth and cardiac function in an in vivo system, immunocompetent female mice were inoculated with cancer cells and treated with the chemotherapeutic drug doxorubicin at a dosing schedule able to suppress tumor growth without inducing cardiac alterations. Analyses carried out in cardiomyocytes treated with the inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), interleukin 6 (IL-6), IL-8, and IL-1β revealed severe phenotypic changes, for example, of contractile cytoskeletal elements, mitochondrial membrane potential, mitochondrial reactive oxygen species production and mitochondria network organization. Accordingly, in immunocompetent mice, the tumor growth was accompanied by increased levels of the inflammatory cytokines TNF-α, IFN-γ, IL-6, and IL-8, either in serum or in the heart tissue, together with a significant reduction of ventricular systolic function. The alterations of mitochondria and of microfilament system of cardiomyocytes, due to the systemic inflammation associated with cancer growth, could be responsible for remote cardiac injury and impairment of systolic function observed in vivo.     

TNF-alpha and H2O2 induce IL-18 and IL-18R beta expression in cardiomyocytes via NF-kappa B activation

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-alpha induced rapid and sustained activation of NF-kappaB whereas H(2)O(2) induced delayed and transient activation. Both TNF-alpha and H(2)O(2) induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-alpha led to sustained expression. Expression of IL-18Rbeta, but not alpha, was induced by both agonists. TNF-alpha and H(2)O(2) induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-alpha and H(2)O(2) induced IL-18 and IL-18Rbeta, but not basal expression of IL-18Ralpha. 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-kappaB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.     

The protective effect of ApolipoproteinA-I on myocardial ischemia-reperfusion injury in rats

It is well established that reperfusion of heart is the optimal method for salvaging ischemic myocardium, however, the success of this therapy could be limited by reperfusion injury, which is involved in inflammatory responses. High density lipoprotein (HDL) has an anti-inflammatory function and can protect the heart from ischemia-reperfusion (I/R) injury. In this study, we investigated the cardioprotective role of apolipoprotein A-I (ApoA-I), the major apolipoprotein of HDL, in I/R injury. Using rats subjected to myocardial I/R by ligation of left anterior descending coronary artery (LAD), we found that administration of ApoA-I (20 mg/kg, iv) before the onset of reperfusion of myocardial infarction can significantly reduce serum creatine kinase (CK) levels (62.1+/-13.8%, p<0.01) and heart TNF-alpha as well as IL-6 levels, compared with saline controls (40.4+/-14.7%, 44+/-9.8%, p<0.01 respectively). Moreover, ApoA-I treatment suppresses the expression of ICAM-1 on endothelium, thus diminishing neutrophil adherence, transendothelial migration, and the subsequent myocyte injury. We concluded that ApoA-I could effectively protect rat heart from I/R injury.     

Toll-like receptor 2 modulates left ventricular function following ischemia-reperfusion injury

Production of proinflammatory cytokines contributes to cardiac dysfunction during ischemia-reperfusion. The principal mechanism responsible for the induction of this innate stress response during periods of myocardial ischemia-reperfusion remains unknown. Toll-like receptor 2 (TLR2) is a highly conserved pattern recognition receptor that has been implicated in the innate immune response to a variety of pathogens. However, TLR2 may also mediate inflammation in response to noninfectious injury. We therefore hypothesized that TLR2 is essential for modulating myocardial inflammation and left ventricular (LV) function during ischemia-reperfusion injury. Susceptibility to myocardial ischemia-reperfusion injury following ischemia-reperfusion was determined in Langendorff-perfused hearts isolated from wild-type mice and mice deficient in TLR2 (TLR2D) and Toll interleukin receptor domain-containing adaptor protein. After ischemia-reperfusion, contractile performance was significantly impaired in hearts from wild-type mice as demonstrated by a lower recovery of LV developed pressure relative to TLR2D hearts. Creatinine kinase levels were similar in both groups after reperfusion. Contractile dysfunction in wild-type hearts was associated with elevated cardiac levels of TNF and IL-1beta. Ischemia-reperfusion-induced LV dysfunction was reversed by treatment with the recombinant TNF blocking protein etanercept. These studies show for the first time that TLR2 signaling importantly contributes to the LV dysfunction that occurs following ischemia-reperfusion. Thus disruption of TLR2-mediated signaling may be helpful to induce immediate or delayed myocardial protection from ischemia-reperfusion injury.     

Systemic inflammation and reperfusion injury in patients with acute myocardial infarction

Despite early recanalization of an occluded infarct artery, tissue reperfusion remains impaired in more than one-third of the acute myocardial infarction (AMI) patients owing to a process of reperfusion injury. The role of systemic inflammation in triggering this phenomenon is unknown. Proinflammatory factors (hs-CRP, TNF-alpha ) and anti-inflammatory mediators (IL-1 receptor antagonist, IL-10) were measured in 65 patients during the acute phase of a myocardial infarction as well as in 11 healthy control subjects. Myocardial reperfusion injury was defined as the presence of persistent ST-segment elevation despite successful coronary intervention (> or = 50 of the initial value) and was observed in 28 patients. Systemic proinflammatory mediators (particularly hs-CRP and leukocytes) were higher in AMI patients compared to control subjects. Within the group of AMI patients, only serum TNF-alpha differed significantly between patients with versus without reperfusion injury: a median value of 25 versus 13 pg/mL was observed, respectively. Logistic regression analysis identified a high level of TNF-alpha as the most important independent determinant of reperfusion injury (P = .001), beyond total ischemic time (P = .01) and extent of jeopardized myocardium (P = .08). There was no correlation between the TNF-alpha level and the total ischemic time (P = .8) or the extent of jeopardized myocardium (P = .6). Systemic inflammation, in particular high levels of TNF-alpha , is strongly associated with the occurrence of reperfusion injury after successful recanalization. Our findings suggest that TNF-alpha is involved in the triggering and/or amplification of local inflammatory responses related to ischemia-reperfusion injury.     

IL-20 promotes hypoxia/reoxygenation-induced mitochondrial dysfunction and apoptosis in cardiomyocytes by upregulating oxidative stress by activating the PKC/NADPH oxidase pathway

Acute myocardial infarction (AMI) is the maximum critical cardiovascular event and causes high morbidity and mortality worldwide. The ischemia and reperfusion that occur in AMI cause apoptosis and cellular dysfunction in cardiomyocytes. IL-20, an IL-10 family member, is involved in various inflammatory diseases. Therefore, we sought to elucidate the role of IL-20 in the infarcted heart following ischemia/reperfusion (I/R) injury. We found that IL-20 and its receptors, IL-20R1 and IL-20R2, were increased in H2C2 cardiomyoblast cells and ventricular tissues subjected to hypoxia/reoxygenation (H/R) stimulation. The presence of IL-20 further inhibited the cell viability of H9C2 cells and primary cardiomyocytes. Our results suggested that IL-20 elicited an increase in Ca²⁺ and activation of the PKC/NADPH oxidase pathway, leading to the elevation of oxidase stress and downregulation of AKT. Furthermore, we demonstrated that IL-20 was able to mediate H/R-induced apoptosis via PKC/NADPH oxidase/AKT signaling. Our findings implied that IL-20 was responsive to H/R stress in vitro and in rat hearts undergoing I/R injury, and this upregulation of IL-20 may contribute to the apoptosis of cardiomyocytes.     

Effects of long-term treatment with eicosapentaenoic acid on the heart subjected to ischemia/reperfusion and hypoxia/reoxygenation in rats

The effects of eicosapentaenoic acid (EPA) and long-term treatment with EPA-ethylester (EPA-E) were examined in perfused rat hearts subjected to ischemia/reperfusion and adult rat cardiomyocytes subjected to hypoxia/reoxygenation. EPA (0.1 M) improved postischmic contractile dysfunction of the ischemic/reperfused heart. EPA (10 M) attenuated hypoxia/reoxygenation-induced morphological deterioration of cardiomyocytes. The results suggest the presence of direct cardioprotective effects of EPA. Rats were orally treated for 4 weeks with 1 g/kg/day of EPA-E to elucidate ex vivo effects of EPA, and the fatty acid composition of cardiac phospholipids was determined. The percent ratio of EPA in total fatty acids of cardiac phospholipids increased whereas that of arachidonic acid decreased. The percent ratio of n-3/n-6 fatty acid did not increase. Treatment with EPA-E did not improve the post-ischemic contractile function, but attenuated the ischemia/reperfusion-induced release of prostaglandins during reperfusion. Treatment with EPA-E preserved a better morphological appearance of the cardiomyocytes subjected to hypoxia/reoxygenation. The results suggest that the mechanisms responsible for cytoprotective effects of hypoxic/reoxygeanted cardiomyocytes or inhibition of metabolic alterations of the ischemic/reperfused heart by long-term EPA-E treatment did not contribute substantially to recovery of post-ischemic contractile dysfunction. The direct in vitro effects of EPA may play a role in the protection of the heart from ischemia/reperfusion or hypoxia/reoxygenation injury.     

Role of interleukin-6 in cardiac inflammation and dysfunction after burn complicated by sepsis

To examine the role of myocardial interleukin-6 (IL-6) in myocardial inflammation and dysfunction after burn complicated by sepsis, we performed 40% total body surface area contact burn followed by late (7 days) Streptococcus pneumoniae pneumonia sepsis in wild-type (WT) mice, IL-6 knockout (IL-6 KO) mice, and transgenic mice overexpressing IL-6 in the myocardium (TG). Twenty-four hours after sepsis was induced, isolated cardiomyocytes were harvested and cultured in vitro, and supernatant concentrations of IL-6 and tumor necrosis factor (TNF)-alpha were measured. Cardiomyocyte intracellular calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) concentrations were also determined. Separate mice in each group underwent in vivo global hemodynamic and cardiac function assessment by cannulation of the carotid artery and insertion of a left ventricular pressure volume conductance catheter. Hearts from these mice were collected for histopathological assessment of inflammatory response, fibrosis, and apoptosis. In the WT group, there was an increase in cardiomyocyte TNF-alpha, [Ca(2+)](i), and [Na(+)](i) after burn plus sepsis, along with cardiac contractile dysfunction, inflammation, and apoptosis. These changes were attenuated in the IL-6 KO group but accentuated in the TG group. We conclude myocardial IL-6 mediates cardiac inflammation and contractile dysfunction after burn plus sepsis.     

Gypenosides alleviate myocardial ischemia-reperfusion injury via attenuation of oxidative stress and preservation of mitochondrial function in rat heart

Gypenosides (GP) are the predominant components of Gynostemma pentaphyllum, a Chinese herb medicine that has been widely used for the treatment of chronic inflammation, hyperlipidemia, and cardiovascular disease. GP has been demonstrated to exert protective effects on the liver and brain against ischemia-reperfusion (I/R) injury, yet whether it is beneficial to the heart during myocardial I/R is unclear. In this study, we demonstrate that pre-treatment with GP dose-dependently limits infarct size, alleviates I/R-induced pathological changes in the myocardium, and preserves left ventricular function in a rat model of cardiac I/R injury. In addition, GP pre-treatment reduces oxidative stress and protects the intracellular antioxidant machinery in the myocardium. Further, we show that the cardioprotective effect of GP is associated with the preservation of mitochondrial function in the cardiomyocytes, as indicated by ATP level, enzymatic activities of complex I, II, and IV on the mitochondrial respiration chain, and the activity of citrate synthase in the citric acid cycle for energy generation. Moreover, GP maintains mitochondrial membrane integrity and inhibits the release of cytochrome c from the mitochondria to the cytosol. The cytoprotective effect of GP is further confirmed in vitro in H9c2 cardiomyoblast cell line with oxygen-glucose deprivation and reperfusion (OGD/R), and the results indicate that GP protects cell viability, reduces oxidative stress, and preserves mitochondrial function. In conclusion, our study suggests that GP may be of clinical value in cytoprotection during acute myocardial infarction and reperfusion.     

The balance between the production of tumor necrosis factor-alpha and interleukin-10 determines tissue injury and lethality during intestinal ischemia and reperfusion

A major goal in the treatment of acute ischemia of a vascular territory is to restore blood flow to normal values, i.e. to "reperfuse" the ischemic vascular bed. However, reperfusion of ischemic tissues is associated with local and systemic leukocyte activation and trafficking, endothelial barrier dysfunction in postcapillary venules, enhanced production of inflammatory mediators and great lethality. This phenomenon has been referred to as "reperfusion injury" and several studies demonstrated that injury is dependent on neutrophil recruitment. Furthermore, ischemia and reperfusion injury is associated with the coordinated activation of a series of cytokines and adhesion molecules. Among the mediators of the inflammatory cascade released, TNF-alpha appears to play an essential role for the reperfusion-associated injury. On the other hand, the release of IL-10 modulates pro-inflammatory cytokine production and reperfusion-associated tissue injury. IL-1beta, PAF and bradykinin are mediators involved in ischemia and reperfusion injury by regulating the balance between TNF-alpha and IL-10 production. Strategies that enhance IL-10 and/or prevent TNF-alpha concentration may be useful as therapeutic adjuvants in the treatment of the tissue injury that follows ischemia and reperfusion.     

Protease Activated Receptor-2 Contributes to Heart Failure

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.     

Local delivery of soluble TNF-alpha receptor 1 gene reduces infarct size following ischemia/reperfusion injury in rats

Apoptosis in the myocardium is linked to ischemia/reperfusion injury, and TNF-alpha induces apoptosis in cardiomyocytes. A significant amount of TNF-alpha is detected after ischemia and reperfusion. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNF-alpha receptor 1 and is an antagonist to TNF-alpha. In the present study, we examined the effects of sTNFR1 on infarct size in acute myocardial infarction (AMI) following ischemia/reperfusion. Male Wistar rats were subjected to left coronary artery (LCA) ligation. After 30 min of LCA occlusion, the temporary ligature on the LCA was released and blood flow was restored. Immediately after reperfusion, a total of 200 microg of sTNFR1 or LacZ plasmid was injected into three different sites of the left ventricular wall. At 6 h, 1 and 2 days after reperfusion, the TNF-alpha bioactivity in the myocardium was significantly higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase in the TNF-alpha bioactivity. The sTNFR1 plasmid significantly reduced DNA fragmentation and caspase activity compared to the LacZ plasmid. Finally, the sTNFR1 expression-plasmid treatment significantly reduced the area of myocardial infarction at 2 days after ischemia/reperfusion compared to LacZ plasmid. In conclusion, the TNF-alpha bioactivity in the heart increased from the early stage of ischemia/reperfusion, and this increase was thought to contribute in part to the increased area of myocardial infarction. Suppression of TNF-alpha bioactivity with the sTNFR1 plasmid reduced the infarct size in AMI following ischemia and reperfusion.     

Therapeutic hypothermia alleviates myocardial ischaemia–reperfusion injury by inhibiting inflammation and fibrosis via the mediation of the SIRT3/NLRP3 signalling pathway

Therapeutic hypothermia (TH) may attenuate myocardial ischaemia–reperfusion injury, thereby improving outcomes in acute myocardial infarction. However, the specific mechanism by which TH alleviates MIRI has not been elucidated so far. In this study, 120 healthy male Sprague‐Dawley rats were randomly divided into five groups. Haemodynamic parameters, myocardial infarction area, histological changes and the levels of cardiac enzymes, caspase‐1 and inflammatory cytokines were determined. In addition, the extent of myocardial fibrosis, the degree of cardiomyocyte apoptosis and the expression levels of SIRT3, GSDMD‐N, fibrosis‐related proteins and inflammation‐related proteins were estimated.TH reduced myocardial infarct area and cardiac enzyme levels, improved cardiomyopathic damage and haemodynamic indexes, and attenuated myocardial fibrosis, the protein expression levels of collagen I and III, myocardial apoptosis, the levels of inflammatory cytokines and inflammation‐related proteins. Notably, the immunofluorescence and protein expression levels of SIRT3 were upregulated in the 34H+DMSO group compared to the I/R group, but this protective effect was abolished by the SIRT3 inhibitor 3‐TYP. After administration of Mcc950, the reversal effects of 3‐TYP were significantly abolished, and TH could protect against MIRI in a rat isolated heart model by inhibiting inflammation and fibrosis. The SIRT3/NLRP3 signalling pathway is one of the most important signalling pathways in this regard.     

LOX-1 pathway affects the extent of myocardial ischemia-reperfusion injury

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was originally identified as a receptor for oxidized low-density lipoprotein predominantly expressed in endothelial cells. LOX-1 expression can be induced in cardiomyocytes and that activation of LOX-1 is involved in apoptosis. To investigate possible roles of LOX-1 in myocardial ischemia-reperfusion injury, rats were subjected to coronary artery ligation for 1h followed by reperfusion for 2h. Immunohistochemistry revealed that expression of LOX-1 in cardiac myocytes was induced following ischemia-reperfusion but not ischemia alone. Administration of anti-LOX-1 monoclonal antibody resulted in a nearly 50% reduction in myocardial infarction size compared with that of normal IgG or saline (P<0.05). These findings suggest that activation of the LOX-1 pathway is involved in determining the extent of myocardial ischemia-reperfusion injury and that inhibition of the LOX-1 pathway may provide a novel strategy for treatment of acute myocardial infarction in humans.