Jesus G. Vallejo

In Vivo Expression of Proinflammatory Mediators in the Adult Heart after Endotoxin Administration: The Role of Toll-Like Receptor–4

Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and nitric oxide (NO) may play a role in lipopolysaccharide (LPS)-induced cardiac depression. Toll-like receptor-4 (TLR-4) mediates the cytokine response to LPS in immune cells. TLR-4 also is expressed in human and murine myocardial tissue. Therefore, the hypothesis that LPS induces proinflammatory cytokines in the heart via TLR-4 was tested. C3H/HeJ (TLR-4 deficient) and C3HeB/FeJ mice were studied. LPS induced a robust increase in myocardial TNF-alpha and IL-1beta mRNA in C3HeB/FeJ mice. The response in C3H/HeJ mice was blunted and delayed. Myocardial TNF-alpha and IL-1beta protein levels were higher in C3HeB/FeJ mice, as were inducible NO synthase protein and NO production. Activation of myocardial NF-kappaB was observed within 30 min in C3HeB/FeJ mice but not in C3H/HeJ mice. These findings suggest that myocardial TLR-4 is involved in signaling cytokine production within the heart during endotoxic shock.

Nuclear Factor-κB Protects the Adult Cardiac Myocyte Against Ischemia-Induced Apoptosis in a Murine Model of Acute Myocardial Infarction

Background—Previous studies have shown that tumor necrosis factor (TNF) confers cytoprotective responses in cardiac myocytes. However, the mechanisms for the cytoprotective effects of TNF remain unknown. Given that TNF signals through nuclear factor &kgr;B (NF-&kgr;B) and given that NF-&kgr;B mediates cytoprotective responses, we asked whether NF-&kgr;B activation conferred cytoprotective responses in acute myocardial ischemia/infarction. Methods and Results—We examined infarct size and the prevalence of apoptosis in transgenic mice harboring cardiac-restricted expression of a mutated I&kgr;B&agr; protein (I&kgr;B&agr;&Dgr;N) that prevents nuclear translocation of NF-&kgr;B in cardiac myocytes. Triphenyltetrazolium chloride staining showed that infarct size was ≈50% greater (P <0.02) in the I&kgr;B&agr;&Dgr;N mice compared with littermate controls at 24 hours. The prevalence of cardiac myocyte apoptosis was significantly greater (P <0.008) in the I&kgr;B&agr;&Dgr;N mice compared with the littermate control mice 3 and 6 hours after left anterior descending occlusion. To explore the mechanism for these findings, we examined protein levels of c-IAP1, c-IAP2, and Bcl-2 as well as manganese superoxide dismutase and c-Jun NH2-terminal kinase activity. These studies showed that protein levels of c-IAP1 and Bcl-2 were significantly lower in the I&kgr;B&agr;&Dgr;N mice, whereas there was no change in c-IAP2 levels, manganese superoxide dismutase, or c-Jun NH2-terminal kinase activity. Conclusions—Transgenic mice with a defect in activation of NF-&kgr;B have increased susceptibility to tissue injury after acute left anterior descending occlusion. These studies suggest that the cytoprotective effects of NF-&kgr;B are mediated, at least in part, by Bcl-2 or c-IAP1.

A Role for Toll-like Receptor 3 Variants in Host Susceptibility to Enteroviral Myocarditis and Dilated Cardiomyopathy

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3β (MAP1LC3β). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3β fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.

CD14-Deficient Mice Are Protected Against Lipopolysaccharide-Induced Cardiac Inflammation and Left Ventricular Dysfunction

Background—The molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined. CD14 mediates the inflammatory response to lipopolysaccharide (LPS) in various organs including the heart. In this study we investigated the role of CD14 in LPS-induced myocardial dysfunction in vivo. Methods and Results—Wild-type and CD14-deficient (CD14-D) mice were challenged with Escherichia coli LPS. Myocardial tumor necrosis factor, interleukin-1&bgr; (IL-1&bgr;), and NOS2 induction was measured before and 6 hours after LPS challenge. Echocardiographic parameters of left ventricular function were measured before and 6 hours after LPS administration. LPS challenge induced a significant increase in myocardial tumor necrosis factor and IL-1&bgr; mRNA and protein expression in wild-type mice. In contrast, mRNA and protein levels for TNF and IL-1&bgr; were significantly blunted in CD14-D mice. An increase in NOS2 protein was noted within 6 hours of LPS provocation only in the hearts of wild-type mice. This was associated with an increase in ventricular cGMP levels. Activation of nuclear factor-&kgr;B was observed within 30 minutes of LPS in the hearts of wild-type mice but not in CD14-D mice. In wild-type mice, LPS significantly decreased left ventricular fractional shortening, velocity of circumferential shortening, and dP/dtmax. LPS-treated CD14-D mice maintained normal cardiac function. Conclusions—These results suggest that CD14 is important in mediating the proinflammatory response induced by LPS in the heart and that CD14 is necessary for the development of left ventricular dysfunction during LPS-induced shock in vivo.

Toll-Like Receptor 2 Mediates Staphylococcus aureus–Induced Myocardial Dysfunction and Cytokine Production in the Heart

Background—Staphylococcus aureus sepsis is associated with significant myocardial dysfunction. Toll-like receptor 2 (TLR2) mediates the inflammatory response to S aureus and may trigger an innate immune response in the heart. We hypothesized that a TLR2 deficiency would attenuate S aureus–induced cardiac proinflammatory mediator production and the development of cardiac dysfunction. Methods and Results—Wild-type and TLR2-deficient (TLR2D) mice were studied. S aureus challenge significantly increased tumor necrosis factor, interleukin-1&bgr;, and nitric oxide expression in hearts of wild-type mice. This response was significantly blunted in TLR2D mice. Hearts from TLR2D mice had impaired S aureus–induced activation of interleukin-1 receptor–associated kinase, c-Jun NH2 terminal kinase, nuclear factor-&kgr;B, and activator protein-1. Moreover, hearts from TLR2D mice were protected against S aureus–induced contractile dysfunction. Conclusions—These results show for the first time that TLR2 signaling contributes to the loss of myocardial contractility and cytokine production in the heart during S aureus sepsis.

Role of Toll-like receptors in cardiovascular diseases

The discovery and characterization of the TLR (Toll-like receptor) family has led to a better understanding of the innate immune system. The strategy of innate immune recognition is based on the detection of constitutive and conserved products of micro-organisms. However, host molecules that are released during injury can also activate TLRs. Engagement of TLRs by microbial or host-derived molecules induces the expression of pro-inflammatory cytokines, which may have both beneficial and detrimental effects on the host. In addition to being expressed in immune cells, TLRs are expressed in other tissues such as those of the cardiovascular system. In the present review, the role of TLRs in septic cardiomyopathy, viral myocarditis, atherosclerosis, ischaemia/reperfusion injury and cardiac remodelling after myocardial infarction are outlined, with attention paid to genetically modified murine models. Although much has been learned about stress-induced TLR activation in the tissues of the cardiovascular system, the role of individual TLRs in initiating and integrating homoeostatic responses within the heart remains to be defined. Accumulating evidence indicates that TLRs may play an important role in the pathogenesis of atherosclerosis, viral myocarditis, dilated cardiomyopathy, cardiac allograft rejection and sepsis-induced left ventricular dysfunction. Moreover, heart failure of diverse aetiology is also now recognized to have an important immune component, with TLR signalling influencing the process of cardiac remodelling and prognosis. In the present review, we outline the biology of TLRs as well as the current experimental and clinical evidence for the role of TLRs in cardiovascular diseases.

Atherogenic diet‐induced hepatitis is partially dependent on murine TLR4

Diets high in cholesterol and cholate such as the Paigen diet have been used to study atherogenesis, lithogenesis, and proinflammatory microvascular changes induced by nutritional hypercholesterolemia. Although these diets lead to chronic hepatic inflammation and fibrosis, the early inflammatory changes have been poorly characterized. TLR4, a known receptor for LPS, is also a receptor for a variety of endogenous ligands and has been implicated in atheroma formation. Here, we specifically examined the early inflammatory response of the liver to the atherogenic (ATH) diet and the possible contribution of TLR4. Animals fed the high‐cholesterol/cholate diet for 3 weeks developed a significant, predominantly mononuclear leukocyte infiltration in the liver, hepatic steatosis, elevated hepatic expression of MCP‐1, RANTES, and MIP‐2, and increased serum levels of liver enzymes. In TLR4‐deleted animals, there was a 30% attenuation in the serum alanine transaminase levels and a 50% reduction in the leukocyte infiltration with a fourfold reduction in chemokine expression. In contrast, hepatic steatosis did not differ from wild‐type controls. TLR2 deletion had no effect on diet‐induced hepatitis but increased the amount of steatosis. We conclude that the early inflammatory liver injury but not hepatic lipid loading induced by the ATH diet in mice is mediated in part by TLR4.

Proapoptotic Effects of Caspase-1/Interleukin-Converting Enzyme Dominate in Myocardial Ischemia

Caspase-1/interleukin-converting enzyme (ICE) is a cysteine protease traditionally considered to have importance as an inflammatory mediator, but not as an apoptotic effector. Because of the dual functions of this caspase, the pathophysiological impact of its reported upregulation in hypertrophy and heart failure is not known. Here, the consequences of increased myocardial expression of procaspase-1 were examined on the normal and ischemically injured heart. In unstressed mouse hearts with a 30-fold increase in procaspase-1 content, unprocessed procaspase-1 was well tolerated, without detectable pathology. Cardiomyocyte processing and activation of caspase-1 and caspase-3 occurred after administration of endotoxin or with transient myocardial ischemia. In post-ischemic hearts, procaspase-1 overexpression was associated with strikingly increased cardiac myocyte apoptosis in the peri- and noninfarct regions and with 50% larger myocardial infarctions. Tissue culture studies revealed that procaspase-1 processing/activation is stimulated by hypoxia, and that caspase-1 acts in synergy with hypoxia to stimulate caspase-3 mediated apoptosis without activating upstream caspases. These data demonstrate that the proapoptotic effects of caspase-1 can significantly impact the myocardial response to ischemia and suggest that conditions in which procaspase-1 in the heart is increased may predispose to apoptotic myocardial injury under conditions of physiological stress.

Toll-like Receptor Signaling Pathways in Cardiovascular Diseases: Challenges and Opportunities

Toll-like receptors (TLRs), a family of surface molecules, are involved in innate immune responses. Recent studies indicated that TLRs play a critical role in inflammatory responses to exogenous and endogenous triggers. This article focuses on probable effects of TLRs in the morbidity of cardiovascular events, e.g., ischemic reperfusion (I/R) injury and atherosclerosis. TLR2 and TLR4 have been shown to have the most fundamental role in promoting cytokine production and subsequent inflammatory damages in these states. Blockade of these receptors may be beneficial in both preventing the occurrence and decreasing the complications in cardiovascular events. However, controversies exist on the certainty of this beneficial effect; therefore, additional studies are needed.

Negative inotropic effects of high-mobility group box 1 protein in isolated contracting cardiac myocytes

High-mobility group box 1 (HMGB1) released from necrotic cells or macrophages functions as a late inflammatory mediator and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients by using fluo-3, and L-type calcium currents by using whole cell perforate configuration of the patch-clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca2+ transient within 5 min (P < 0.01). The immediate negative inotropic effects of HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward l-type calcium currents was also documented by the patch-clamp technique. HMGB1 induced the PKC-epsilon translocation, and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx and suggest that HMGB1 maybe acts as a novel myocardial depressant factor during cardiac injury.