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Wnt signaling is dysregulated in heart failure (HF) and may promote cardiac hypertrophy, fibrosis, and inflammation. Blocking the Wnt ligand Wnt5a prevents HF in animal models. However, the role of Wnt5a in human HF and its functions in cardiac cells remain unclear. Here, we investigated Wnt5a regulation in HF patients and its effects on primary mouse and human cardiac fibroblasts. Serum Wnt5a was elevated in HF patients and associated with hemodynamic, neurohormonal, and clinical measures of disease severity. In failing human hearts, Wnt5a protein correlated with interleukin (IL)-6 and tissue inhibitor of metalloproteinase (TIMP)-1. Wnt5a messenger RNA (mRNA) levels were markedly upregulated in failing myocardium and both mRNA and protein levels declined following left ventricular assist device therapy. In primary mouse and human cardiac fibroblasts, recombinant Wnt5a dose-dependently upregulated mRNA and protein release of IL-6 and TIMP-1. Wnt5a did not affect β-catenin levels, but activated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Importantly, inhibition of ERK1/2 activation attenuated Wnt5a-induced release of IL-6 and TIMP-1. In conclusion, our results show that Wnt5a is elevated in the serum and myocardium of HF patients and is associated with measures of progressive HF. Wnt5a induces IL-6 and TIMP-1 in cardiac fibroblasts, which might promote myocardial inflammation and fibrosis, and thereby contribute to HF progression. • Wnt5a is elevated in serum and myocardium of HF patients and is associated with measures of progressive HF. • In cardiac fibroblasts, Wnt5a upregulates interleukin (IL)-6 and tissue inhibitor of metalloproteinase (TIMP)-1 through the ERK pathway. • Wnt5a-mediated effects might promote myocardial inflammation and fibrosis, and thereby contribute to HF progression.

An inflammatory response is required for tissue healing after a myocardial infarction (MI), but the process must be balanced to prevent maladaptive remodeling. This study shows that improved survival and cardiac function following MI, in mice deficient for the NLRP3 inflammasome, can be recapitulated in wild-type mice receiving bone marrow from Nlrp3−/− mice. This suggests that NLRP3 activation in hematopoietic cells infiltrating in the myocardium increases mortality and late ventricular remodeling. Our data should encourage performing clinical trials directly targeting NLRP3 inflammasome and their inflammatory cytokines (interleukin-1β and -18) in MI patients. SEVIER ON BEHALF OF THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER THE CC BY-NC-ND LICENSE ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) .

Background The innate immune receptor NLRP3 recognizes tissue damage and initiates inflammatory processes through formation multiprotein complexes with the adaptor protein ASC and caspase-1, i.e. NLRP3 inflammasomes, which through cleavage of pro-IL-1β mediates release of bioactive IL-1β. We hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction (MI) and sought to investigate the mechanisms herein in an experimental MI model in mice. Methods and results The left coronary artery (LCA) of WT, NLRP3 -/- and ASC -/- mice of both genders was ligated for 30 min followed by 3 or 24 h reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3CSK4 or PBS was injected intraperitoneally 60 min prior to LCA ligation. For mechanistic investigations, blood plasmas and left ventricle tissues were collected, and a hypothesis-driven selection of protein or mRNA targets was investigated. Surprisingly, hearts from NLRP3-deficient mice featured larger infarct size than WT mice (p = 0.0048). In general, there were only modest changes with no significant pattern in myocardial infiltration of neutrophils and macrophages and systemic and myocardial cytokine expression between the three genotypes. Preconditioning with the TLR2 agonist Pam3CSK4 induced Akt phosphorylation and reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. Conclusion Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardioprotective RISK pathway. Our data imply that NLRP3 contributes to cardio-protection during I/R and do not support a role for NLRP3 or ASC inhibition in the management of acute MI including revascularization therapy.

Myocardial infarction (MI) triggers a reparative response involving fibroblast proliferation and differentiation driving extracellular matrix modulation necessary to form a stabilizing scar. Recently, it was shown that a genetic variant of the base excision repair enzyme NEIL3 was associated with increased risk of MI in humans. Here, we report elevated myocardial NEIL3 expression in heart failure patients and marked myocardial upregulation of Neil3 after MI in mice, especially in a fibroblast-enriched cell fraction. Neil3−/− mice show increased mortality after MI caused by myocardial rupture. Genome-wide analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) reveals changes in the cardiac epigenome, including in genes related to the post-MI transcriptional response. Differentially methylated genes are enriched in pathways related to proliferation and myofibroblast differentiation. Accordingly, Neil3−/− ruptured hearts show increased proliferation of fibroblasts and myofibroblasts. We propose that NEIL3-dependent modulation of DNA methylation regulates cardiac fibroblast proliferation and thereby affects extracellular matrix modulation after MI. © 2017 The Authors. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision.Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups.Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3-/- and ASC-/-mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1β, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated β-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

Aim Myocardial infarction (MI) remains a major cause of death and disability worldwide, despite available reperfusion therapies. Inflammatory signaling is considered nodal in defining final infarct size. Activation of the innate immune receptor toll-like receptors (TLR) 9 prior to ischemia and reperfusion (I/R) reduces infarct size, but the consequence of TLR9 activation timed to the onset of ischemia is not known. Methods and results The TLR9-agonist; CpG B was injected i.p. in C57BL/6 mice immediately after induction of ischemia (30 minutes). Final infarct size, as well as area-at-risk, was measured after 24 hours of reperfusion. CpG B injection resulted in a significant increase in circulating granulocytes and monocytes both in sham and I/R mice. Paradoxically, clear evidence of reduced cardiac infiltration of both monocytes and granulocytes could be demonstrated in I/R mice treated with CpG B (immunocytochemistry, myeloperoxidase activity and mRNA expression patterns). In addition, systemic TLR9 activation elicited significant alterations of cardiac inflammatory genes. Despite these biochemical and cellular changes, there was no difference in infarct size between vehicle and CpG B treated I/R mice. Conclusion Systemic TLR9-stimulation upon onset of ischemia and subsequent reperfusion does not alter final infarct size despite causing clear alterations of both systemic and cardiac inflammatory parameters. Our results question the clinical usefulness of TLR9 activation during cardiac I/R.

Aims N ucleotide-binding oligomerization domain- L ike R eceptor with a P yrin domain 3 (NLRP3) is considered necessary for initiating a profound sterile inflammatory response. NLRP3 forms multi-protein complexes with A poptosis-associated S peck-like protein containing a C aspase recruitment domain (ASC) and Caspase-1, which activate pro-interleukin-1β (IL-1β) and pro-IL-18. The role of NLRP3 in cardiac cells is not known. Thus, we investigated the expression and function of NLRP3 during myocardial ischaemia. Methods and results Myocardial infarction (MI) was induced in adult C57BL/6 mice and Wistar rats by ligation of the coronary artery. A marked increase in NLRP3, IL-1β, and IL-18 mRNA expression was found in the left ventricle after MI, primarily located to myocardial fibroblasts. In vitro studies in cells from adult mice showed that myocardial fibroblasts released IL-1β and IL-18 when primed with lipopolysaccharide and subsequently exposed to the danger signal adenosine triphosphate, a molecule released after tissue damage during MI. When hearts were isolated from NLRP3-deficient mice, perfused and subjected to global ischaemia and reperfusion, a marked improvement of cardiac function and reduction of hypoxic damage was found compared with wild-type hearts. This was not observed in ASC-deficient hearts, potentially reflecting a protective role of other ASC-dependent inflammasomes or inflammasome-independent effects of NLRP3. Conclusion This study shows that the NLRP3 inflammasome is up-regulated in myocardial fibroblasts post-MI, and may be a significant contributor to infarct size development during ischaemia–reperfusion.

Purpose: Type 2 diabetes (T2D) is an important risk factor for heart failure. T2D is associated with the metabolic syndrome, encompassing cardiovascular risk factors such as obesity, insulin resistance and dyslipidemia, factors also associated with left ventricular diastolic dysfunction. Patients with T2D have elevated plasma levels of free fatty acids and these are proposed to activate inflammation. Interleukin-1β (IL-1β) is activated by the NLRP3 inflammasome and a central mediator in T2D. Palmitate, the most abundant saturated fatty acid in plasma was recently shown to activate the NLRP3 inflammasome in macrophages. The aim of this study was to investigate if this mechanism operates within myocardial cells. Methods and Results: We first replicated previous findings by incubating bone marrow-derived macrophages with palmitate, and found release of active IL-1β. We went on to study primary mouse cardiac fibroblasts (CF). CF were stimulated with LPS, causing priming of the inflammasome, and then with palmitate. This caused release of active IL-1β. Importantly, this was not found in CF from NLRP3 and ASC deficient mice, demonstrating that the effect of palmitate on IL-1β release is mediated through the NLRP3 inflammasome. Other fatty acids such as eicosapentaenoic acid (EPA), oleic acid (OA), docosahexaenoic acid did not cause inflammasome activation. The omega-3 fatty EPA is known to suppress palmitate-induced metabolic stress, and when combining palmitate and EPA, we found markedly reduced release of IL-1β. By viewing CF stained with Bodipy under fluorescence microscopy, we observed an inhibitory effect of EPA on PA-induced lipid accumulation. AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis and also suppresses reactive oxygen species (ROS) production. We found that palmitate markedly reduced AMPK phosphorylation and Mitosox staining revealed increased mitochondrial ROS. Addition of the pharmacological AMPK activator AICAR completely blocked palmitate-induced IL-1β production. AMPK is also involved in regulation of autophagy, and we found that palmitate reduced levels of beclin-1, indicating that reduced autophagy could represent a mechanism for palmitate-induced inflammasome activation. Conclusion: Palmitate causes activation of the NLRP3 inflammasome in cardiac fibroblasts and this may involve increased mitochondrial ROS and defective autophagy. These findings indicate that NLRP3 inflammasome may act as a sensor of metabolic stress in the heart. EPA inhibits PA-induced NLRP3 inflammasome activation, possibly via reduced lipid accumulation.

Purpose: Immune activation plays a nodal role in the pathogenesis of heart failure (HF) and Toll-like receptor 9 (TLR9) signaling significantly impacts the development of systolic HF. However, the consequence of altered TLR9 activity in diastolic HF is unknown. Methods: We engaged in a 3-generation breeding strategy using αMHC-MerCreMer Serca2a flox/flox mice crossed with TLR9-/- mice to generate several comparable mouse lines. Employing these various genetic mouse lines we addressed the importance of TLR9 on progression of diastolic HF, the latter by temporally restricted depletion of cardiac myocyte SERCA2a. Mice were allocated to two substudies; 1) a 12 week survival study registering spontaneous death or signs of severe morbidity according to pre-specified criteria (leading to euthanasia) and 2) a study examining mice at baseline, 3 weeks and 6 weeks by MRI and echocardiography. After imaging at 6 weeks mice were euthanized and tissue and blood were harvested. Results: All mice depleted of cardiomyocyte SERCA2a, but none with the SERCA2a gene intact, reached our pre-specified end-parameter within 73 days. The presence of TLR9 in this model of diastolic HF led to significant improvement in survival with a median life expectancy of 62.5 days as compared to 58 days when the TLR9 gene was deleted (p=0.007). In accordance with these findings serial imaging demonstrated an earlier onset of left ventricular restrictive filling abnormalities in the HF group depleted of TLR9. Significantly larger left atria, lower end-systolic and end-diastolic left ventricular volumes and lower stroke volumes could be detected. Conclusions: The presence of TLR9 augments the development of diastolic HF induced by SERCA2a depletion. This observation may pave the road for novel diastolic HF treatment strategies. ![Figure][1] Survival in days after HF induction [1]: pending:yes

Purpose: Systemic inflammation in established HF is clinically relevant as many HF patients have concomitant chronic inflammatory conditions. Repetitive activation of Toll-like receptor 9 (TLR9) induces chronic inflammation. We used a pharmacological protocol to induce systemic, chronic inflammation in murine cardiomyopathy induced by cardiomyocyte specific deletion of SERCA2a. Methods: Cardiac expression of SERCA2a was deleted in 10 week old male αMHC-MerCreMer Serca2a flox/flox mice by one intraperitoneal injection of Tamoxifen, resulting in a lethal diastolic HF phenotype after 7 weeks. 4 weeks after conditional knock out (KO), mice were randomized to TLR9 agonist (CpG-B;50 mg i.p.) or PBS every 3 days. Echocardiography was performed 8 weeks after induction of HF, followed by euthanization and analyses of tissue and blood. The groups were followed for 12 weeks, registering death and morbidity (by pre-specified criteria). Non-HF control groups treated with CpG-B or PBS served as controls. Results: TLR9 stimulated mice showed alterations in levels of leukocytes, erythrocytes and platelets as measured by flow cytometry and peripheral blood count, as well as pulmonary inflammation and hypersplenism (histology). The alterations were not found in controls. Echocardiographic analyses revealed aggravation in diastolic parameters in HF mice treated with CpG B. The latter group had reduced life expectancy compared with HF controls (median 59.5 and 68.5 days respectively, p=0.003). Non-HF, CpG B treated mice did not die or display signs of disease within the observation period. Conclusions: Chronic inflammation promoted by continuous TLR9 stimulation worsens diastolic HF caused by SERCA2a deletion. This study may provide insight into the consequences of chronic inflammatory conditions on HF development. ![Figure][1] Survival in days after HF induction [1]: pending:yes

Background: NLRP3 is an innate immune receptor that recognizes tissue damage and initiate inflammatory processes: chiefly, upon activation, NLRP3 forms multiprotein complexes with the adaptor protein ASC and caspase-1, termed inflammasomes, which cleaves pro-IL-1β and mediates release of bioactive IL-1β. Thus, NLRP3 activation may lead to further tissue damage. Hence, we hypothesized that NLRP3 mediates tissue damage during acute myocardial infarction and sought to investigate the mechanisms herein. Methods: The LAD of WT, NLRP3 KO and ASC KO mice of both genders were sham-operated or ligated in vivo for 30 minutes followed by 3 or 24 hours reperfusion. For pre-conditioning studies, the TLR2 agonist Pam3Cys or PBS was injected intraperitoneally 60 minutes prior to LAD ligation. In animals subjected to 24 hours reperfusion, the LAD was re-ligated in vivo and non-ischemic tissue stained by retrograde perfusion of Evans Blue in the aorta. The area at risk was stained after slicing with TTC, revealing the area of non-stained tissue as the infarct size. For further mechanistic investigations, blood plasmas and left ventricle tissues were collected and a hypothesis-driven selection of protein or mRNA targets were investigated with Western blot, ELISA, bioplex or PCR. Results: No difference in the area at risk was observed between any groups. Surprisingly, hearts from NLRP3 -and ASC deficient mice featured larger infarct size than that of WT mice (p=0.0048 and not statistically significant [p=0.0840], respectively). Furthermore, preconditioning with the TLR2 agonist Pam3Cys reduced infarct size in WT but not NLRP3 -or ASC -deficient hearts. There was no difference in plasma troponin I, IL-1β or IL-1α between any groups at 3 or 24 hours. In accordance with this, there were similar corresponding mRNA levels in cardiac tissue. However, there were significantly higher levels of CXCL1 in plasma from NLRP3-deficient mice at 3 hours. Preconditioning with Pam3Cys and MI for 30 min, resulted in increased phosphorylation of the cardioprotective kinase AKT in WT mice. However, this was not observed in NLRP3 -or ASC deficient mice, indicating a role for the NLRP3 inflammasome in cardioprotection. Conclusion: Absence of NLRP3 results in increased myocardial infarct size after in vivo ischemia reperfusion, seemingly due to dysfunction of the cardio-protective p-AKT signalling pathway involved in preconditioning. Furthermore, increased CXCL1 chemokine release may result in more tissue damage due to recruitment of neutrophils. Hence, our data imply that NRLP3 contribute to cardio-protection during ischemia-reperfusion.

Accumulation of DNA damage due to impaired base excision repair (BER) may play a role in the pathogenesis of myocardial failure. The DNA glycosylase Neil3 is known to carry out BER during oxidative stress induced DNA damage, but is also thought to have a role in stem cell biology. Based on this, we hypothesized that Neil3 plays a role in myocardial remodeling during heart failure (HF). qPCR showed an increased expression of Neil3 in myocardial biopsies from patients with severe HF, and that the Neil3 expression significantly decreased following improvement of myocardial function following treatment with left ventricular assist device. In a murine model of myocardial infarction (MI) we found a strong upregulation of Neil3 on day 3, 7 and 21 after MI. At day 7, myocardial cell isolation revealed a low Neil3 expression in cardiomyocytes, while a 2.5- and a 4.5-fold increased expression was found in endothelial cells and fibroblasts, respectively, following MI compared to sham. In leukocytes an equally high expression of Neil3 was observed in both sham and MI. Using Neil3 knock-out (KO) mice we found a significantly higher mortality in KO compared to wild type (WT) mice after MI ( 73% vs. 40%), despite smaller infarct sizes in the Neil3 KO, as determined by MRI. The first week post MI, all animals died from cardiac rupture. Zymography on protein extracts from the infarcted tissue revealed significantly increased levels of MMP2 in the Neil3 KO, 3 days after MI. Immunohistochemistry (IHC), at the same time point, showed similar numbers of inflammatory cells measured by the presence of Ly6G+ neutrophils, Mac2+ monocytes/macrophages and CD45+ leukocytes. At day 6 a significantly higher number of Ki67+ proliferating cells were observed in the Neil3 KO infarcted areas. In accordance with this, primary cardiac fibroblasts lacking Neil3 showed a higher proliferation rate, measured both by BrdU and scrape wound assay. Furthermore, we found a significantly higher mRNA expression of both the myofibroblast marker α-SMA, and collagen type III in cardiac fibroblasts isolated from Neil3 KO mice 7 days post MI, indicating increased levels of activated fibroblasts and altered fibroblast function. IHC 6 days post MI showed the same tendency towards more α-SMA-positive fibroblasts and more collagen by Sirius red in the Neil3 KO infarcts, however these differences were not statistically significant. In conclusion, our data suggest that after MI, Neil3 plays a role in regulating fibroblast proliferation and function, affecting wound healing and extracellular matrix composition and quality, altogether reducing mortality.