P619Role of Toll-like receptor 5 in the development of post-myocardial infarction inflammation

Background: Inflammatory processes play a key role in the pathophysiology of myocardial infarction (MI). Genetic deletion of toll-like recpetors (TLRs), especially TLR2 and TLR4 have shown protective role in murine models of MI. The role of other TLRs remains unknown. We have previously shown that cardiomyocytes express TLR5 and that the ligand of TLR5, flagellin, activates the NF-kappaB and MAPK pathways in cardiomyocytes. We also have shown that injection of flagellin induces acute systolic dysfunction in vivo in mice. Aim: Determine the role of TLR5 in the development of post-MI inflammation. Methods: A murine model of myocardial infarction was done by a 30 minutes ligation of the left anterior descending coronary artery followed by 2 hours of reperfusion. Infarct size was measured by standard Evans blue/TTC staining. Plasma creatine kinase (CK) was quantified as a read out of myocardial necrosis. Tissue and plasma cytokines (MIP-2, MCP-1, IL-6) were quantified by ELISA. To determine the extent of tissue lipid peroxidation we used malondialdehyde and 4-hydroxynonenal-HIS adduct assays. Tissue protein oxidation was tested by protein carbonyl ELISA kit. Phosphorylation of MAPK was analyzed by western blot. Results: Genetic suppression of TLR5 induced a significant increase of myocardial infarct size and plasma CK, of biochemical markers of myocardial oxidative stress, and cytokine levels in the heart and the plasma after MI. These effects were associated with a marked enhancement of p38 phosphorylation in the heart from TLR5 KO mice. Conclusion: TLR5 protects from acute myocardial injury and reduces local and systemic inflammation during myocardial infarction. The mechanisms may involve reduced p38 signaling, decreased oxidative stress and attenuated cytokine expression. Research supported by the Swiss National Science Foundation, Grant n° 310030_135394/

P90Necrotic cardiomyocytes release soluble pro-inflammatory molecule(s) inducing il1r/myd88-dependent inflammatory responses in cardiac fibroblasts

Background: Inflammation comes out to be a critical biological process in the pathophysiology of myocardial infarction (MI). We hypothesize that this inflammation is triggered by necrotic cardiomyocytes (Cmc) that release a set of endogenous molecules (DAMPs: danger-associated molecular patterns) activating inflammatory responses in cardiac fibroblasts. Aim: Analyze in vitro the immune activation of cardiac fibroblasts exposed to necrotic Cmc conditioned media. Methods: Primary neonatal murine cardiac fibroblasts and Cmc were obtained by digestion of neonatal hearts and differential plating technique allowing a selection for cardiomyocytes and cardiac fibroblasts. Cmc were killed by necrotic stimuli including oxidants (hydrogen peroxide) and mechanic stresses (freeze-thaw). Necrosis was assessed using Hoechst/PI stainings. Fibroblasts were exposed to necrotic Cmc conditioned media and mRNA expression of inflammatory genes was measured by real-time PCR and ELISA. Activation of signaling pathways was analyzed by western blot. We used cardiac cells from Myd88-/-, Trif-/- and Nlrp3-/- animals to evaluate the contribution of TLRs/IL1-R and NLRP3 inflammasome in the sensing of necrotic DAMPs. Results: mRNA expression of chemokines such as MCP-1, MIP-2 and IP-10 were induced in fibroblasts exposed to necrotic Cmc conditioned media. Alternatively, fibroblasts exposed to necrotic fibroblasts conditioned media showed a lower increase in mRNA expression of these chemokines. In addition, in fibroblasts from Myd88-/- mice, response to Cmc conditioned media was fully abrogated whereas no difference was observed in Trif-/- and Nlrp3-/- fibroblasts. Conclusion: Cardiac fibroblasts are able to produce a rapid and specific inflammatory response to necrotic Cmc conditioned media involving the expression of neutrophil and monocyte chemoattractants. The dependence on MyD88 adaptor protein strongly suggests that this response relies on TLR/IL-1R signaling. These results engage cardiac fibroblasts as key players in post-MI inflammatory responses as they are able to sense DAMPs from necrotic Cmc and possibly recruit inflammatory cells. Research supported by the Swiss National Science Foundation, Grant n° 310030_135394/

Natriuretic Peptide Receptor B modulates the proliferation of the cardiac cells expressing the Stem Cell Antigen-1.

Brain Natriuretic Peptide (BNP) injections in adult "healthy" or infarcted mice led to increased number of non-myocyte cells (NMCs) expressing the nuclear transcription factor Nkx2.5. The aim of this study was to identify the nature of the cells able to respond to BNP as well as the signaling pathway involved. BNP treatment of neonatal mouse NMCs stimulated Sca-1 <sup>+</sup> cell proliferation. The Sca-1 <sup>+</sup> cells were characterized as being a mixed cell population involving fibroblasts and multipotent precursor cells. Thus, BNP treatment led also to increased number of Sca-1 <sup>+</sup> cells expressing Nkx2.5, in Sca-1 <sup>+</sup> cell cultures in vitro and in vivo, in the hearts of neonatal and adult infarcted mice. Whereas BNP induced Sca-1 <sup>+</sup> cell proliferation via NPR-B receptor and protein kinase G activation, CNP stimulated Sca-1 <sup>+</sup> cell proliferation via NPR-B and a PKG-independent mechanism. We highlighted here a new role for the natriuretic peptide receptor B which was identified as a target able to modulate the proliferation of the Sca-1 <sup>+</sup> cells. The involvement of NPR-B signaling in heart regeneration has, however, to be further investigated

The role of oxidative stress during inflammatory processes.

Abstract The production of various reactive oxidant species in excess of endogenous antioxidant defense mechanisms promotes the development of a state of oxidative stress, with significant biological consequences. In recent years, evidence has emerged that oxidative stress plays a crucial role in the development and perpetuation of inflammation, and thus contributes to the pathophysiology of a number of debilitating illnesses, such as cardiovascular diseases, diabetes, cancer, or neurodegenerative processes. Oxidants affect all stages of the inflammatory response, including the release by damaged tissues of molecules acting as endogenous danger signals, their sensing by innate immune receptors from the Toll-like (TLRs) and the NOD-like (NLRs) families, and the activation of signaling pathways initiating the adaptive cellular response to such signals. In this article, after summarizing the basic aspects of redox biology and inflammation, we review in detail the current knowledge on the fundamental connections between oxidative stress and inflammatory processes, with a special emphasis on the danger molecule high-mobility group box-1, the TLRs, the NLRP-3 receptor, and the inflammasome, as well as the transcription factor nuclear factor-κB

Impact of aerobic exercise type on blood flow, muscle energy metabolism, and mitochondrial biogenesis in experimental lower extremity artery disease.

Exercise training (ET) is recommended for lower extremity artery disease (LEAD) management. However, there is still little information on the hemodynamic and metabolic adaptations by skeletal muscle with ET. We examined whether hindlimb perfusion/vascularization and muscle energy metabolism are altered differently by three types of aerobic ET. ApoE <sup>-/-</sup> mice with LEAD were assigned to one of four groups for 4 weeks: sedentary (SED), forced treadmill running (FTR), voluntary wheel running (VWR), or forced swimming (FS). Voluntary exercise capacity was improved and equally as efficient with FTR and VWR, but remained unchanged with FS. Neither ischemic hindlimb perfusion and oxygenation, nor arteriolar density and mRNA expression of arteriogenic-related genes differed between groups. <sup>18</sup> FDG PET imaging revealed no difference in the steady-state levels of phosphorylated <sup>18</sup> FDG in ischemic and non-ischemic hindlimb muscle between groups, nor was glycogen content or mRNA and protein expression of glucose metabolism-related genes in ischemic muscle modified. mRNA (but not protein) expression of lipid metabolism-related genes was upregulated across all exercise groups, particularly by non-ischemic muscle. Markers of mitochondrial content (mitochondrial DNA content and citrate synthase activity) as well as mRNA expression of mitochondrial biogenesis-related genes in muscle were not increased with ET. Contrary to FTR and VWR, swimming was ineffective in improving voluntary exercise capacity. The underlying hindlimb hemodynamics or muscle energy metabolism are unable to explain the benefits of running exercise

Cutting edge: IL-1α is a crucial danger signal triggering acute myocardial inflammation during myocardial infarction.

Myocardial infarction (MI) induces a sterile inflammatory response that contributes to adverse cardiac remodeling. The initiating mechanisms of this response remain incompletely defined. We found that necrotic cardiomyocytes released a heat-labile proinflammatory signal activating MAPKs and NF-κB in cardiac fibroblasts, with secondary production of cytokines. This response was abolished in Myd88(-/-) fibroblasts but was unaffected in nlrp3-deficient fibroblasts. Despite MyD88 dependency, the response was TLR independent, as explored in TLR reporter cells, pointing to a contribution of the IL-1 pathway. Indeed, necrotic cardiomyocytes released IL-1α, but not IL-1β, and the immune activation of cardiac fibroblasts was abrogated by an IL-1R antagonist and an IL-1α-blocking Ab. Moreover, immune responses triggered by necrotic Il1a(-/-) cardiomyocytes were markedly reduced. In vivo, mice exposed to MI released IL-1α in the plasma, and postischemic inflammation was attenuated in Il1a(-/-) mice. Thus, our findings identify IL-1α as a crucial early danger signal triggering post-MI inflammation

Exenatide Improves Glucose Homeostasis and Prolongs Survival in a Murine Model of Dilated Cardiomyopathy

There is growing awareness of secondary insulin resistance and alterations in myocardial glucose utilization in congestive heart failure. Whether therapies that directly target these changes would be beneficial is unclear. We previously demonstrated that acute blockade of the insulin responsive facilitative glucose transporter GLUT4 precipitates acute decompensated heart failure in mice with advanced dilated cardiomyopathy. Our current objective was to determine whether pharmacologic enhancement of insulin sensitivity and myocardial glucose uptake preserves cardiac function and survival in the setting of primary heart failure.The GLP-1 agonist exenatide was administered twice daily to a murine model of dilated cardiomyopathy (TG9) starting at 56 days of life. TG9 mice develop congestive heart failure and secondary insulin resistance in a highly predictable manner with death by 12 weeks of age. Glucose homeostasis was assessed by measuring glucose tolerance at 8 and 10 weeks and tissue 2-deoxyglucose uptake at 75 days. Exenatide treatment improved glucose tolerance, myocardial GLUT4 expression and 2-deoxyglucose uptake, cardiac contractility, and survival over control vehicle-treated TG9 mice. Phosphorylation of AMP kinase and AKT was also increased in exenatide-treated animals. Total myocardial GLUT1 levels were not different between groups. Exenatide also abrogated the detrimental effect of the GLUT4 antagonist ritonavir on survival in TG9 mice.In heart failure secondary insulin resistance is maladaptive and myocardial glucose uptake is suboptimal. An incretin-based therapy, which addresses these changes, appears beneficial

Assessment of Metabolic Phenotypes in Patients with Non-ischemic Dilated Cardiomyopathy Undergoing Cardiac Resynchronization Therapy

Studies of myocardial metabolism have reported that contractile performance at a given myocardial oxygen consumption (MVO2) can be lower when the heart is oxidizing fatty acids rather than glucose or lactate. The objective of this study is to assess the prognostic value of myocardial metabolic phenotypes in identifying non-responders among non-ischemic dilated cardiomyopathy (NIDCM) patients undergoing cardiac resynchronization therapy (CRT). Arterial and coronary sinus plasma concentrations of oxygen, glucose, lactate, pyruvate, free fatty acids (FFA), and 22 amino acids were obtained from 19 male and 2 female patients (mean age 56 ± 16) with NIDCM undergoing CRT. Metabolite fluxes/MVO2 and extraction fractions were calculated. Flux balance analysis (FBA) was performed with MetaFluxNet 1.8 on a metabolic network of the cardiac mitochondria (189 reactions, 230 metabolites) reconstructed from mitochondrial proteomic data (615 proteins) from human heart tissue. Non-responders based on left ventricular ejection fraction (LVEF) demonstrated a greater mean FFA extraction fraction (35% ± 17%) than responders [18 ± 10%, p = 0.0098, area under the estimated ROC curve (AUC) was 0.8238, S.E. 0.1115]. Calculated adenosine triphosphate (ATP)/MVO2 using FBA correlated with change in New York Heart Association (NYHA) class (rho = 0.63, p = 0.0298; AUC = 0.8381, S.E. 0.1316). Non-responders based on both LVEF and NYHA demonstrated a greater mean FFA uptake/MVO2 (0.115 ± 0.112) than responders (0.034 ± 0.030, p = 0.0171; AUC = 0.8593, S.E. 0.0965). Myocardial FFA flux and calculated maximal ATP synthesis flux using FBA may be helpful as biomarkers in identifying non-responders among NIDCM patients undergoing CRT

Adenovirus-mediated stromal cell-derived factor-1 alpha gene transfer improves cardiac structure and function after experimental myocardial infarction through angiogenic and antifibrotic actions

Stromal cell-derived factor 1α (SDF-1) is not only a major chemotactic factor, but also an inducer of angiogenesis. The effects of SDF-1α on the left ventricular remodeling in a rat myocardial infarction (MI) model were analyzed. Myocardial infarction was induced by ligation of the left coronary artery in rats. 0.5 × 1010 pfu/ml AdV-SDF-1 or 0.5 × 1010 pfu/ml Adv-LacZ were immediately injected into the infarcted myocardium, 120 μl cell-free PBS were injected into the infarcted region or the myocardial wall in control, and sham group, respectively. We found that AdV-SDF-1 group had higher LVSP and ±dP/dtmax, lower LVEDP compared to control or Adv-LacZ group. The number of c-Kit+ stem cells, and gene expression of SDF-1, VEGF and bFGF were obviously increased, which was associated with reduced infarct size, thicker left ventricle wall, greater vascular density and cardiocytes density in infarcted hearts of AdV-SDF-1 group. Furthermore, the expression of collagen type I and type III mRNA, and collagen accumulation in the infarcted area was lower, which was associated with decreased TGF-β1, TIMP-1 and TIMP-2 expression in AdV-SDF-1 group. Conclusion: SDF-1α could improve cardiac structure and function after Myocardial infarction through angiogenic and anti-fibrotic actions