Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts.

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-1ß, 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 ß-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.

Toll-Like Receptor 9 Promotes Survival in SERCA2a KO Heart Failure Mice.

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.

Low Circulating Levels of Mitochondrial and High Levels of Nuclear DNA Predict Mortality in Chronic Heart Failure.

BACKGROUND: Mitochondrial DNA (mtDNA) and possibly nuclear DNA (nDNA) are released as danger-associated molecular patterns during cardiac stress, and may activate several innate immune receptors. The purpose of this study was to investigate the regulation of these danger-associated molecular patterns during human heart failure (HF). METHODS AND RESULTS: Plasma levels of mtDNA and nDNA from HF patients (n = 84) were analyzed by reverse transcriptase-polymerase chain reaction and compared with controls (n = 72). Increased levels of mtDNA were found in New York Heart Association (NYHA) I-II and NYHA III-IV. There was evidence of increased nDNA in NYHA III-IV compared with controls and NYHA I-II. Kaplan-Meier analysis revealed higher mortality in patients with high nDNA levels, whereas high levels of mtDNA were associated with survival. CONCLUSIONS: Plasma levels of mtDNA and nDNA are elevated in human HF associated with increased and decreased mortality, respectively. This study may suggest a rationale for exploring interventions within inflammatory signaling pathways activated by nucleic acids as novel targets in treatment of HF.

Collagen isoform shift during the early phase of reverse left ventricular remodelling after relief of pressure overload.

AIMS: Aortic stenosis induces pressure overload and myocardial remodelling with concentric hypertrophy and alterations in extracellular matrix (ECM). Aortic valve replacement leads to reverse remodelling, a process of which knowledge is scarce. The aims of the present study were to examine alterations in myocardial gene expression and subsequently identify molecular alterations important for the early phase of reverse remodelling. METHODS AND RESULTS: After 4 weeks of ascending aortic banding, mice were subjected to a debanding operation (DB) and followed for 3, 7, or 14 days. Cardiac function was assessed by echocardiography/tissue Doppler ultrasonography. Myocardial gene expression was examined using Affymetrix microarray and the topGO software and verified by real-time polymerase chain reaction. Quantitative measurements of collagen subtypes were performed. Aortic banding increased left ventricular mass by 60%, with normalization to sham level 14 days after DB. Extracellular matrix genes were the most regulated after DB. Three days after DB, collagen I was transiently increased, whereas collagens III and VIII increased later at 7 days. CONCLUSION: The ECM genes were the most altered during reverse remodelling. There was a change in isoform constitution as collagen type I increased transiently at 3 days followed by a later increase in types III and VIII at 7 days after DB. This might be important for the biomechanical properties of the heart and recovery of cardiac function.

Moderate heart dysfunction in mice with inducible cardiomyocyte-specific excision of the Serca2 gene.

The sarco(endo)plasmic reticulum calcium ATPase 2 (SERCA2) transports Ca(2+) from cytosol into the sarcoplasmic reticulum (SR) of cardiomyocytes, thereby maintaining the store of releasable Ca(2+) necessary for contraction. Reduced SERCA function has been linked to heart failure, and loss of SERCA2 in the adult mammalian heart would be expected to cause immediate severe myocardial contractile dysfunction and death. We investigated heart function in adult mice with an inducible cardiomyocyte-specific excision of the Atp2a2 (Serca2) gene (SERCA2 KO). Seven weeks after induction of Serca2 gene excision, the mice displayed a substantial reduction in diastolic function with a 5-fold increase in the time constant of isovolumetric pressure decay (tau). However, already at 4 weeks following gene excision less than 5% SERCA2 protein was found in myocardial tissue. Surprisingly, heart function was only moderately impaired at this time point. Tissue Doppler imaging showed slightly reduced peak systolic tissue velocity and a less than 2-fold increase in tau was observed. The SR Ca(2+) content was dramatically reduced in cardiomyocytes from 4-week SERCA2 KO mice, and Ca(2+) transients were predominantly generated by enhanced Ca(2+) flux through L-type Ca(2+) channels and the Na(+)-Ca(2+) exchanger. Moreover, equivalent increases in cytosolic [Ca(2+)] in control and SERCA2 KO myocytes induced greater cell shortening in SERCA2 KO, suggesting enhanced myofilament responsiveness. Our data demonstrate that SR-independent Ca(2+) transport mechanisms temporarily can prevent major cardiac dysfunction despite a major reduction of SERCA2 in cardiomyocytes.

Cytokine expression profiling of the myocardium reveals a role for CX3CL1 (fractalkine) in heart failure.

Several lines of evidence suggest that inflammatory processes mediated by cytokines are involved in the pathogenesis of heart failure (HF). However, the regulation of cytokine expression and the role of cytokines during HF development are not well understood. To address this issue, we have examined alterations in gene expression during HF progression by microarray technology in non-infarcted left ventricular (LV) murine tissue at various time points after myocardial infarction (MI). The highest number of regulated genes was found five days after MI. In total, we identified 14 regulated genes encoding cytokines with no previous association to HF. The strongest up-regulation was found for the chemokine fractalkine (CX3CL1). In human failing hearts we detected a 3-fold increase in CX3CL1 protein production, and both cardiomyocytes and fibrous tissue revealed immunoreactivity for CX3CL1 and its specific receptor CX3CR1. We also found that the circulating level of CX3CL1 was increased in patients with chronic HF in accordance with disease severity (1.6-fold in NYHA II, 2.2-fold in NYHA III and 2.9-fold in NYHA IV). In vitro experiments demonstrated that CX3CL1 production could be induced by inflammatory cytokines known to be highly expressed in HF. CX3CL1 itself induced the expression of markers of cardiac hypertrophy and protein phosphatases in neonatal cardiomyocytes. Given the increased CX3CL1 production in both an experimental HF model and in patients with chronic HF as well as its direct effects on cardiomyocytes, we suggest a role for CX3CL1 and its receptor CX3CR1 in the pathogenesis of HF.

Echocardiographic parameters discriminating myocardial infarction with pulmonary congestion from myocardial infarction without congestion in the mouse.

Our aim was to establish parameters describing systolic and diastolic function in mice after myocardial infarction (MI) that distinguish MI with pulmonary congestion from MI without congestion. Echocardiography, left ventricular (LV) catheterization, and infarct size measurements were performed on days 3, 5, 7, and 14 after ligation of the left coronary artery in C57BL/6 mice. Sham-operated mice were used as controls (Sham). MI mice with lung weight normalized to tibial length >125% of the average in the corresponding Sham group were considered to have pulmonary congestion (MIchf). MI mice with a smaller increase were called MI nonfailing (MInf). An infarct >40% of total LV circumference measured in two-dimensional long axis distinguished MIchf from MInf on both an average and an individual basis. Mean maximum rate of rise of LV pressure, LV fractional shortening, and posterior wall shortening velocity were significantly lower in MIchf compared with Sham at all time points and to MInf at 7 days. The diastolic parameters mitral flow deceleration velocity, LV end-diastolic pressure, and maximum rate of decline in LV pressure (LVdP/dtmin) discriminated the MIchf groups from Sham at all time points. Mitral flow deceleration velocity and LVdP/dtmin separated MIchf from MInf at 7 days. In addition to distinguishing all the groups on an average basis, left atrial diameter distinguished all MIchf animals from Sham and MInf. In conclusion, significantly increased left atrial diameter and infarct size >40% of total LV circumference may serve as major criteria for heart failure with pulmonary congestion after MI in mice.

Increased syndecan expression following myocardial infarction indicates a role in cardiac remodeling.

The purpose of this study was to identify essential genes involved in myocardial growth and remodeling following myocardial infarction (MI). Left ventricular noninfarcted tissues from six mice subjected to MI under general anesthesia and from six sham-operated mice were obtained 1 wk after primary surgery and analyzed by means of cDNA filter arrays. Out of a total of 1,176 genes, 641 were consistently expressed, twenty-three were upregulated and thirteen downregulated. Five genes were only expressed following MI. Syndecan-3, a transmembranous heparan sulfate proteoglycan, was found to be upregulated together with a transcriptional activator of syndecans, Wilms tumor protein 1 (WT-1). Northern blotting demonstrated a significant upregulation of syndecan-1, -2, -3, and -4, WT-1, fibronectin, and basic fibroblast growth factor (FGF) receptor 1. Furthermore, Western blot analysis showed statistically significant increases in protein levels for syndecan-3 and -4. In conclusion, we have identified a subset of genes with increased expression in noninfarcted left ventricular tissue following MI, including syndecans 1-4, WT-1, fibronectin, collagen 6A, and FGF receptor 1. Since the syndecans link the cytoskeleton to the extracellular matrix and function as required coreceptors for FGF, we suggest a role for the syndecans in cardiac remodeling following MI.

Toll-like receptor 9 mediated responses in cardiac fibroblasts.

Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/ß). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

The homeostatic chemokine CCL21 predicts mortality in aortic stenosis patients and modulates left ventricular remodeling.

BACKGROUND: CCL21 acting through CCR7, is termed a homeostatic chemokine. Based on its role in concerting immunological responses and its proposed involvement in tissue remodeling, we hypothesized that this chemokine could play a role in myocardial remodeling during left ventricular (LV) pressure overload. METHODS AND RESULTS: Our main findings were: (i) Serum levels of CCL21 were markedly raised in patients with symptomatic aortic stenosis (AS, n = 136) as compared with healthy controls (n = 20). (ii) A CCL21 level in the highest tertile was independently associated with all-cause mortality in these patients. (iii) Immunostaining suggested the presence of CCR7 on macrophages, endothelial cells and fibroblasts within calcified human aortic valves. (iv). Mice exposed to LV pressure overload showed enhanced myocardial expression of CCL21 and CCR7 mRNA, and increased CCL21 protein levels. (v) CCR7-/- mice subjected to three weeks of LV pressure overload had similar heart weights compared to wild type mice, but increased LV dilatation and reduced wall thickness. CONCLUSIONS: Our studies, combining experiments in clinical and experimental LV pressure overload, suggest that CCL21/CCR7 interactions might be involved in the response to pressure overload secondary to AS.

The homeostatic chemokine CCL21 predicts mortality and may play a pathogenic role in heart failure.

BACKGROUND: CCL19 and CCL21, acting through CCR7, are termed homeostatic chemokines. Based on their role in concerting immunological responses and their proposed involvement in tissue remodeling, we hypothesized that these chemokines could play a pathogenic role in heart failure (HF). METHODOLOGY/PRINCIPAL FINDINGS: Our main findings were: (i) Serum levels of CCL19 and particularly CCL21 were markedly raised in patients with chronic HF (n = 150) as compared with healthy controls (n = 20). A CCL21 level above median was independently associated with all-cause mortality. (ii) In patients with HF following acute myocardial infarction (MI; n = 232), high versus low CCL21 levels 1 month post-MI were associated with cardiovascular mortality, even after adjustment for established risk factors. (iii). Explanted failing human LV tissue (n = 29) had markedly increased expression of CCL21 as compared with non-failing myocardium (n = 5). (iv) Our studies in CCR7(-/-) mice showed improved survival and attenuated increase in markers of myocardial dysfunction and wall stress in post-MI HF after 1 week, accompanied by increased myocardial expression of markers of regulatory T cells. (v) Six weeks post-MI, there was an increase in markers of myocardial dysfunction and wall stress in CCR7 deficient mice. CONCLUSIONS/SIGNIFICANCE: High serum levels of CCL21 are independently associated with mortality in chronic and acute post-MI HF. Our findings in CCR7 deficient mice may suggest that CCL21 is not only a marker, but also a mediator of myocardial failure. However, while short term inhibition of CCR7 may be beneficial following MI, a total lack of CCR7 during long-term follow-up could be harmful.

Chromogranin B in heart failure: a putative cardiac biomarker expressed in the failing myocardium.

BACKGROUND: Chromogranin B (CgB) is a member of the granin protein family. Because CgB is often colocalized with chromogranin A (CgA), a recently discovered cardiac biomarker, we hypothesized that CgB is regulated during heart failure (HF) development. METHODS AND RESULTS: CgB regulation was investigated in patients with chronic HF and in a post-myocardial infarction HF mouse model. Animals were phenotypically characterized by echocardiography and euthanized 1 week after myocardial infarction. CgB mRNA levels were 5.2-fold increased in the noninfarcted part of the left ventricle of HF animals compared with sham-operated animals (P<0.001). CgB mRNA level in HF animals correlated closely with animal lung weight (r=0.74, P=0.04) but not with CgA mRNA levels (r=0.20, P=0.61). CgB protein levels were markedly increased in both the noninfarcted (110%) and the infarcted part of the left ventricle (70%) but unaltered in other tissues investigated. Myocardial CgB immunoreactivity was confined to cardiomyocytes. Norepinephrine, angiotensin II, and transforming growth factor-beta increased CgB gene expression in cardiomyocytes. Circulating CgB levels were increased in HF animals (median levels in HF animals versus sham, 1.23 [interquartile range, 1.03 to 1.93] versus 0.98 [0.90 to 1.04] nmol/L; P=0.003) and in HF patients (HF patients versus control, 1.66 [1.48 to 1.85] versus 1.47 [1.39 to 1.58] nmol/L; P=0.007), with levels increasing in proportion to New York Heart Association functional class (P=0.03 for trend). Circulating CgB levels were only modestly correlated with CgA (r=0.31, P=0.009) and B-type natriuretic peptide levels (r=0.27, P=0.014). CONCLUSIONS: CgB production is increased and regulated in proportion to disease severity in the left ventricle and circulation during HF development.