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The importance of cytoskeleton in neutrophil-cardiomyocyte interaction

University of British Columbia

Activated polymorphonuclear neutrophils (PMN) contribute to decreased myocardial contractility after ischemia-reperfusion and in models of sepsis. To determine how PMN could contribute to myocardial dysfunction, rat ventricular cardiomyocytes were isolated and incubated with TNFα 20 ng/mL, IL-1β 20 ng/mL or LPS 10 pg/mL for 3 hours. Cardiomyocytes were then washed, co-cultured with isolated rat PMN for 2 hours, and electrically stimulated to determine fractional shortening (FS) using videomicroscopy. PMN co-cultured with activated cardiomyocytes reduced fractional shortening by 30±10% (p<0.001). Fractional shortening of cardiomyocytes with adherent PMN decreased by - 2:8±0.3% per adherent PMN (p<0.001). Fixing PMN with paraformaldehyde or gluteraldehyde did not prevent PMN-mediated decreases in cardiomyocyte fractional shortening. PMN adherence and the PMN-mediated decrease in fractional shortening were prevented by anti-ICAM-1 and anti-CD18 antibodies. Reduced fractional shortening by 36 ± 3% was reproduced in the absence of PMN by ICAM-1 binding using cross-linking antibodies. To determine whether this reduction in fractional shortening could be through coupling of ICAM-1 receptors to the cardiomyocyte cytoskeleton, we disrupted F-actin filament assembly by pre-incubating cardiomyocytes for with cytochalasin D (10 μM) or latrunculin A (10 μM) respectively for 2 hours. These compounds did not prevent PMN adherence but significantly reduced the effect of adherent PMN on cardiomyocyte contractility. Latruculin A and Cytochalasin D also reduced the effects of ICAM-1 cross-linking mediated decrease in cardiomyocyte contractility. Inhibiting MEK1 using PD98059 had no effect while inhibition of RhoA using HA-1077 prevented the ICAM-1 mediated decrease in cardiomyocyte contractility. Immunofluorescent staining demonstrated that ICAM-1 cross linking increased total focal adhesion kinase (FAK) expression in the cortical cytoskeleton. Focal adhesion kinase (FAK) phosphorylation on tyrosin 397 was lower in the 3 hours ICAM-1 cross-linked group compared to control. Erk1/2 and P38 MAPK were not significantly different in the two groups. We conclude that PMN decrease cardiomyocyte contractility by binding to ICAM-1, which mediates part of the decreased contractility through RhoA and the cardiomyocyte cytoskeleton.

Thesis/Dissertation

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2009-08-12

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http://hdl.handle.net/2429/12075

Experimental Medicine

University of British Columbia

UBCV

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