Molecular Mechanisms in Heart Failure : The Role of Syndecan-4 in Development of Myocardial Hypertrophy and Heart Failure

The prevalence of congestive heart failure (CHF) is increasing dramatically world-wide. Sustained myocardial hypertrophy is an important risk factor for developing CHF, but the underlying mechanisms initiating and maintaining hypertrophy are still poorly understood. The aim of this thesis was to identify such molecular mechanisms. We demonstrate a crucial role for the membrane protein syndecan-4 in development of concentric hypertrophy in response to pressure overload, probably due to its activation of the important pro-hypertrophic calcineurin-nuclear factor of activated T cell (NFAT) pathway. In contrast to wild type mice (WT), syndecan-4-/- mice (Syn-4-/-) showed no development of concentric hypertrophy following aortic-banding. The calcineurin-NFAT pathway was specifically inhibited in Syn-4-/-. We were able to demonstrate that syndecan-4 binds calcineurin, and the respective binding domains were identified. Moreover, a short cell-permeable blocking-peptide, specific for the syndecan-4-calcineurin interaction, was generated and significantly inhibited calcineurin-NFAT signaling. We therefore believe that syndecan-4 may represent a novel target for treatment of myocardial hypertrophy and heart failure

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.publishedVersio

Toll-Like Receptor 9 Mediated Responses in Cardiac Fibroblasts

<div><p>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/β). 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 <i>in vitro</i>. Finally, results from <i>in vivo</i> TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our <i>in vitro</i> 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.</p></div

Comparative analysis of TLR9-stimulated responses between cardiac fibroblasts and bone-marrow derived -macrophages and –dendritic cells.

<p>Dose-response relationships of (100 ng/ml) CpG A (panels A–B), CpG B (panels C–D) and CpG C (panels E–F) stimulated release of CXCL2 (left panels) and TNFα (right panels) were examined in murine cardiac fibroblasts (CFs; circles) compared to bone marrow derived –macrophages (triangles) and –dendritic cells (DC; squares). Analysis was performed after 18 hours stimulation. Each data point represents the mean ± SEM of 3 experiments.</p

<i>In vivo</i> cardiac TLR9-stimulated cellular responses.

<p>Male C57BL/6 mice were injected i.p. with 100 µl CpG B (50 µg; n = 6, black bars) or vehicle (n = 6, white bars) and euthanized after 24 hours, with subsequent isolation of cardiac myocytes (CM), CD45⁺, CD31⁺ and non-CM/non-CD45⁺/non-CD31⁺ (denominated CF). TLR9-mediated responses in the cell-fractions were analyzed by mRNA expression levels of CXCL2 (panels A–D) and TNFα (panels E–H). Uncorrected data are shown in panels A and E. CXCL2 and TNFα are also presented as corrected for GAPDH (panels B and F), β-actin (panels C and G) and 18S (panels D and H). Data presented as mean ± SEM. *<i>p</i><0.05 vs. sham, **<i>p</i><0.01 vs. sham.</p

Primer sequences used in qPCR assays.

<p>GAPDH was chosen as housekeeping gene.</p

TLR9-stimulated responses in cardiac fibroblasts.

<p><b>Temporal profiles</b> (10 min, 30 min, 1 h, 2 h, 5 h, 10 h and 24 h) of (all at 100 ng/ml) CpG A (dots), CpG B (open circles) or CpG C (squares)-stimulated release of CXCL2 (panel A) and TNFα (panel B) in murine cardiac fibroblasts (CF). Each data point represents the mean ± SEM of 3 separate experiments. <b>Dose-response relationships</b> of CpG A (dots), CpG B (open circles) and CpG C (squares) –stimulated release of CXCL2 (panel C) and TNFα (panel D) at 18 hours. Each data point represents the mean ± SEM of 6 experiments. Expression levels of CXCL2 (panel E) and TNFα (panel F) were analyzed by qPCR. Each data point represents the mean ± SEM of 4 experiments. <b>TLR9 specificity studies</b> were performed on murine CFs exposed to CpG B (100 ng/ml) or LPS (10 ng/ml) with or without the presence of ODN 2088 (10 µg/ml) or chloroquine diphosphate (5 µg/ml). Levels of CXCL2 (panel G) and TNFα (panel H) were analyzed after 18 hours. Each data point represents the mean ± SEM of 3 experiments.</p

TLR9 mRNA levels in cultivated murine cardiac fibroblasts (CF; n = 3), bone marrow derived macrophages (n = 3) and dendritic cells (DC; n = 3).

<p>Data presented as mean ± SEM.</p