Fibrinogen decreases cardiomyocyte contractility through an ICAM-1-dependent mechanism

Abstract

Introduction: Cardiomyocytes exposed to inflammatory processes express intracellular adhesion molecule-1 (ICAM-1). We investigated whether fibrinogen and fibrinogen degradation products, including D-dimer, could alter cardiomyocyte contractile function through interaction with ICAM-1 found on inflamed cardiomyocytes.

Methods: In vivo, rats were injected with endotoxin to model systemic inflammation, whereas isolated rat cardiomyocytes were treated with tumor necrosis factor-alpha to model the inflammatory environment seen following exposure to bacterial products such as lipopolysaccharide.

Results: In vivo, endotoxin administration profoundly decreased cardiac contractile function associated with a large increase in intracardiac ICAM-1 and perivascular fibrinogen. Confocal microscopy with double-staining of isolated rat cardiomyocytes demonstrated colocalization of ICAM-1 and fibrinogen. This interaction was disrupted through pre-treatment of the cells with an ICAM-1-blocking antibody. Functionally, isolated rat cardiomyocyte preparations exhibited decreased fractional shortening when incubated with fibrinogen, and through the use of synthetic peptides, we determined that residues 117-133 of the fibrinogen gamma chain are responsible for this interaction with ICAM-1. Despite having crosslinked gamma chains, D-dimer retained the ability to decrease cardiomyocyte contractility.

Conclusion: Site 117-133 of the fibrinogen gamma chain is able to depress cardiomyocyte contractility through binding ICAM-1.

Figure 1

LPS decreases cardiac contractility. Cardiac cycle pressure-volume loops obtained 6 hours after intraperitoneal injection of lipopolysaccharide (LPS) or saline into rats. Acquired with group mean data, the curves demonstrate an LPS-induced increased end diastolic volume (EDV), maintenance of stroke volume (SV), and therefore a marked reduction of left ventricular ejection fraction (SV/EDV). E_es, end-systolic elastance.

Figure 2

LPS increases intracardiac ICAM-1 and perivascular fibrinogen. (a) Frozen cardiac sections from lipopolysaccharide (LPS)-treated and saline-treated rats demonstrate that intracardiac intracellular adhesion molecule-1 (ICAM-1) (red) is dramatically increased in the former. (b) Fibrinogen (green) was greatly increased outside the endothelium (von Willebrand factor labeled red) in the LPS group compared with rats treated with saline. (c) Group mean data of the fold increases in myocardial ICAM-1 expression and perivascular fibrinogen deposition in LPS-treated versus saline-treated animals. *p < 0.05 versus saline.

Figure 3

Fibrinogen binds specifically to cardiomyocyte ICAM-1. Colocalization of fibrinogen and cardiomyocyte intracellular adhesion molecule-1 (ICAM-1). (a) Isolated cardiomyocytes were incubated with Oregon Green-labeled fibrinogen (green) and fluorescently stained for ICAM-1 (red). A multiphoton dual-excitation image of the contour of the cell of interest is shown. By means of an overlay of images, strong colocalization of fibrinogen and ICAM-1 was indicated by a yellow color. (b) A representative image of a rat cardiomyocyte with adherent fibrinogen-coated polystyrene beads (white arrows) is shown to the left of the graph. The specificity of the ICAM-1-fibrinogen interaction is demonstrated as anti-ICAM-1 antibody pre-treatment results in significantly less fibrinogen-coated polystyrene beads adherent to the cardiomyocytes (*p < 0.05 versus control). Ab, antibody; CL, control; IgG, immunoglobulin G.

Figure 4

ICAM-1 (8–22) binds to fibrinogen. Two-step enzyme-linked immunosorbent assay in which fibrinogen was immobilized on the 96-well plate and then incubated with biotinylated intracellular adhesion molecule-1 (ICAM-1) (8–22) peptide or biotinylated 'scrambled' control peptide. Anti-biotin antibody was added, and colorimetric absorbance quantified the peptide-fibrinogen interaction. (a) Representative ICAM-1 (8–22) dose-response curve showing absorbance plateau at a fibrinogen concentration of approximately 100 μM. (b) Group mean absorbance data taken at the plateau fibrinogen concentration (100 μM) demonstrating a strong interaction between the ICAM-1 (8–22) peptide and fibrinogen compared with a small non-specific interaction between the scrambled peptide and fibrinogen. *p < 0.05 versus scrambled peptide.

Figure 5

Fibrinogen decreases cardiomyocyte fractional shortening via ICAM-1. Cardiomyocytes were pre-treated with either a blocking anti-ICAM-1 antibody or isotype control antibody prior to the addition of fibrinogen-coated beads. Fractional shortening was then measured. Whereas pre-treatment with immunoglobulin G (IgG) isotype antibody was no different than fibrinogen alone, treatment with blocking anti-ICAM-1 antibody prevented the fibrinogen-induced decrease in fractional shortening. *p < 0.05 versus fibrinogen beads alone. Ab, antibody; FBG, fibrinogen; ICAM-1, intracellular adhesion molecule-1.

Figure 6

ICAM-1 (8–22) mediates decreased contractility. Before fibrinogen was added to activated cardiomyocytes, soluble intracellular adhesion molecule-1 (ICAM-1) (8–22) peptide, which binds fibrinogen at peptides 117–133, is added in excess to the fibrinogen. Activated cardiomyocytes incubated with fibrinogen alone demonstrate a 40% reduction in contractility. Pre-incubation of fibrinogen with the ICAM-1 (8–22) peptide results in competition between cardiomyocyte-expressed ICAM-1 and the ICAM-1 (8–22) peptide for the fibrinogen active site (117–133). Pre-incubation with the ICAM-1 (8–22) peptide abolishes the reduced contractility seen with fibrinogen alone, whereas pre-incubation with 'scrambled' ICAM-1 peptide had no effect. *p < 0.05 versus control. Fbg, fibrinogen.

Figure 7

Fibrinogen subunit D decreases contractility. (a) Schematic diagram of the fibrinogen molecule, showing two D chains each containing a gamma chain linked to a central E chain. (b) Cardiomyocytes were incubated with whole fibrinogen as well as the major subunits D and E of fibrinogen. Whole fibrinogen and subunit D resulted in significant decreases in fractional shortening (FS), whereas subunit E had no significant effect. *,^†p < 0.05 versus control.

Figure 8

Fibrinogen gamma chain and D-dimer decrease contractility. (a) Schematic diagram of D-dimer with two D subunits linked in part via interaction of the gamma chains. For simplicity, we have not shown the areas on the D chain itself which participate in dimerization. (b) Expanded diagram of the gamma subunit showing the crosslinking (XL) site of the C-terminal regions which results in amine donor lysine 406 of one gamma chain and a glutamine acceptor at residue 398 or 399. The intracellular adhesion molecule-1 (ICAM-1) binding site is shown as residue 117–133, far removed from the XL site. (c) Cardiomyocytes were incubated with a peptide with the sequence 117–133, D-dimer, or scrambled peptide. D-dimer and the gamma (117–133) peptide resulted in significant decreases in fractional shortening (FS), whereas scrambled peptide had no significant effect. *,^†p < 0.05 versus control.