Progress in Biophysics and Molecular Biology
ReviewActions of emigrated neutrophils on Na+ and K+ currents in rat ventricular myocytes
Introduction
Inflammation within the ischaemic myocardium is a complex process. Numerous immunological and cell signalling mechanisms contribute to tissue damage. Alone or in combination, these can result in reduced myocardial function. Recently, attention has focussed on the deleterious effects of macrophages in inflamed myocardium (Entman et al., 1992; Mehta and Li, 1999). Within hours of initiation of an inflammatory response there is an infiltration of myocardial tissues by neutrophils as assessed by both histological changes (Binah, 1994; Bohle et al., 1993; Birdsall et al., 1997) and biochemical markers (Chen et al., 1995; Lefer et al., 1996; Cuevas et al., 1997; Sato et al., 1997). Adhesion of emigrated neutrophils to ventricular myocytes compromises their function and it may result in cell death (Wilson et al., 1993; Lefer et al, 1993; Mayers et al., 1996; Semb et al., 1989).
Neutrophil-mediated target cell responses involve a cascade of distinct events. After tissue injury the production of cytokines and chemoattractants results in the activation of neutrophils and enhanced expression of adhesion molecules (Entman et al., 1992). This is followed by extravation of neutrophils from the circulation to the site(s) of injury. These emigrated neutrophils adhere strongly to myocytes and ultimately cause myocyte dysfunction (Entman et al., 1990; Smith et al., 1991; Poon et al., 1999). Although the process of leukocyte recruitment is now quite well understood (cf. Poon et al., 1999); the neutrophil–myocyte interactions have been studied in less detail. In their important, original studies, Entman et al., 1990, Entman et al., 1992 demonstrated that neutrophils can adhere to myocytes via CD11/CD18 or B2-integrins. Recently, we have identified an additional type of adhesion molecule, α4-integrin, and shown that it is also an important mediator of neutrophil adhesion to myocytes (Reinhardt et al., 1997). Following adhesion to myocytes, the activated neutrophils release a number of cytotoxic compounds, each of which can cause myocyte dysfunction (Rossen et al., 1985; Engler, 1989; Lucchesi et al., 1989) sometimes including arrhythmias (Dhein et al., 1995).
Previous studies have demonstrated that application of a number of the substances known to be released by neutrophils can quickly result in dramatically altered mechanical and electrophysiological responses in cardiac myocytes (Pallandi et al., 1987; Barrington et al., 1988). Hoffman et al (1997) reported that circulating neutrophils, when bound to canine myocytes, produced electrophysiological effects including delayed repolarization, early afterdepolarizations (EAD) and, ultimately, marked depolarization of the resting potential. These results provided important insights into the way in which neutrophils may generate rhythm disturbances, and also suggested that neutrophils may be important contributors to reperfusion-induced arrhythmias. These findings also raised questions concerning the underlying ionic mechanism(s) i.e. whether neutrophil-induced changes in transmembrane ionic currents could be identified. These questions and the known functional differences between activated and emigrated neutrophils (Zimmerli et al., 1986; Yee et al., 1994; Poon et al., 1999) provided the background to and the motivation for these neutrophil/myocyte studies.
The present experiments were designed to record the electrophysiological changes induced following adherence of either circulating or emigrated neutrophils to rat ventricular myocytes, and to identify the corresponding changes in ionic current(s). Our results show that shortly after adherence of emigrated neutrophils, rapid and marked electrophysiological changes (increased action potential duration, depolarization of resting membrane potential) occur. These changes may quickly result in myocyte death. Corresponding voltage clamp measurements demonstrate that neutrophil adherence leads to alterations in the (i) TTX-sensitive Na+ current and (ii) inwardly rectifying K+ current, IK1. When the voltage clamp method was used to prevent neutrophil-mediated membrane depolarization and/or activation of Na+ current, myocyte damage was reduced and the myocyte death increased dramatically. We suggest, therefore, that the primary cytotoxic effects of neutrophils on cardiac myocytes appears to arise from neutrophil-induced changes of TTX-sensitive Na+ channels. These electrophysiological findings have been incorporated into a modified version of our mathematical model for the rat ventricular action potential (Pandit et al., 2001). In this way, our results have been integrated compared with previous literature, and evaluated in terms of a number of different working hypotheses for the underlying electrophysiological and electrochemical changes.
Section snippets
Ventricular cell isolation
The procedure for the isolation of adult rat ventricular myocytes has been described previously by Ward and Giles (1997). Briefly, animals were killed by decapitation and the heart was rapidly removed and mounted on a canula for retrograde perfusion at a rate of 10 ml min−1 using a standard Langendorff apparatus. Hearts were initially perfused for 5 min with Tyrode's solution containing 1 mM CaCl2. This was followed by 5 min perfusion with nominally Ca2+-free Tyrode's solution. After this, the heart
Results
In all experiments, emigrated neutrophils adhered strongly to freshly isolated quiescent ventricular myocytes. Under our experimental conditions, on average, 4.2±0.4 () emigrated neutrophils adhered to each ventricular myocyte. In contrast, when the same density of circulating neutrophils were added to the superfusion solution, only 1.5±1.0 () neutrophils/myocyte adhered. Fig. 1 shows representative microscopic fields of myocytes in the presence of either (A) circulating or (B) emigrated
Discussion
Recent studies have demonstrated the presence of neutrophils in inflamed myocardium, either as a consequence of primary inflammation or following reperfusion of previously ischaemic myocardium (Lefer et al., 1993; Wilson et al., 1993; Sato et al., 1997; Mehta and Li, 1999). Neutrophil-mediated events can give rise to tissue injury and may contribute to the generation of life threatening arrhythmias (Dhein et al., 1995). Previously, we have reported that neutrophils could adhere in a selective,
Acknowledgments
The study was supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada. WRG held an Alberta Heritage Foundation for Medical Research (AHFMR) Medical Scientist Award and was also supported (2004–2005) by the Departments of Bioengineering and Medicine at the University of California San Diego. CAW was supported by Heart and Stroke Foundation of Canada and AHFMR Postdoctoral Fellowships. The financial assistance of the Research Chair of the Heart and
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