Review
Actions of emigrated neutrophils on Na+ and K+ currents in rat ventricular myocytes

https://doi.org/10.1016/j.pbiomolbio.2005.07.003Get rights and content

Abstract

Interactions between neutrophils and the ventricular myocardium can contribute to tissue injury, contractile dysfunction and generation of arrhythmias in acute cardiac inflammation. Many of the molecular events responsible for neutrophil adhesion to ventricular myocytes are well defined; in contrast, the resulting electrophysiological effects and changes in excitation–contraction coupling have not been studied in detail. In the present experiments, rat ventricular myocytes were superfused with either circulating or emigrated neutrophils and whole-cell currents and action potential waveforms were recorded using the nystatin-perforated patch method. Almost immediately after adhering to ventricular myocytes, emigrated neutrophils caused a depolarization of the resting membrane potential and a marked prolongation of myocyte action potential. Voltage clamp experiments demonstrated that following neutrophil adhesion, there was (i) a slowing of the inactivation of a TTX-sensitive Na+ current, and (ii) a decrease in an inwardly rectifying K+ current.

One cytotoxic effect of neutrophils appears to be initiated by enhanced Na+ entry into the myocytes. Thus, manoeuvres that precluded activation of Na+ channels, for example holding the membrane potential at −80 mV, significantly increased the time to cell death or prevented contracture entirely. A mathematical model for the action potential of rat ventricular myocytes has been modified and then utilized to integrate these findings. These simulations demonstrate the marked effects of (50-fold) slowing of the inactivation of 2–4% of the available Na+ channels on action potential duration and the corresponding intracellular Ca2+ transient. In ongoing studies using this combination of approaches, are providing significant new insights into some of the fundamental processes that modulate myocyte damage in acute inflammation.

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 (n=39) 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 (n=4) 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

References (56)

  • I. Baczko et al.

    Resting membrane potential regulates Na+–Ca2+ exchange-mediated Ca2+ overload during hypoxia-reoxygenation in rat ventricular myocytes

    J. Physiol.

    (2003)
  • I. Baczko et al.

    Pharmacological activation of plasma-membrane KATP channels reduces reoxygenation-induced Ca2+ overload in cardiac myocytes via modulation of the diastolic membrane potential

    Br. J. Pharm.

    (2004)
  • O. Binah

    Immune effector mechanism in heart transplant rejection

    Cardiovasc. Res.

    (1994)
  • H.H. Birdsall et al.

    Complement C5a, TGF-beta 1, and MCP-1, in sequence, induce migration of monocytes into ischemic canine myocardium within the first one to five hours after reperfusion

    Circulation

    (1997)
  • M.P. Blaustein et al.

    Sodium/calcium exchange: its physiological implications

    Physiol. Rev.

    (1999)
  • R.M. Bohle et al.

    Interstitial myocardial neutrophil accumulation between 3 and 72 h of reperfusion does not significantly affect infarct size in porcine hearts

    Am. J. Cardiovasc. Pathol.

    (1993)
  • V.E. Bondarenko et al.

    Computer model of action potential of mouse ventricular myocytes

    Am. J. Physiol. Heart Circ. Physiol.

    (2004)
  • R.A. Bouchard et al.

    Role of sodium-calcium exchange in activation of contraction in rat ventricle

    J. Physiol. (London)

    (1993)
  • R.B. Bouchard et al.

    Action potential voltage-clamp measurements of intracellular calcium homeostasis and E–C coupling in rat ventricular myocytes

    Circ. Res.

    (1995)
  • R. Bouchard et al.

    Changes in extracellular K+ concentration modulate contractility of rat and rabbit cardiac myocytes via the inward rectifier K+ current IK1

    J. Physiol. (London)

    (2004)
  • D.L. Campbell et al.

    The cardiac calcium-independent outward potassium current: kinetics, molecular properties, and role in ventricular repolarization

  • E. Cerbai et al.

    Cellular electrophysiological basis for oxygen radical-induced arrhythmias. A patch clamp study in guinea pig ventricular myocytes.

    Circulation

    (1991)
  • C.E. Clancy et al.

    Linking a genetic defect to its cellular phenotype in a cardiac arrhythmia

    Nature

    (1999)
  • R.B. Clark et al.

    Action potential duration modulates calcium influx Na+–Ca2+ exchange, and intracellular calcium release in rat ventricular myocytes

    Ann. N.Y. Acad. Sci.

    (1996)
  • P. Cuevas et al.

    A non-mitogenic form of acidic fibroblast growth factor reduces neutrophil infiltration in rat ischemic reperfused heart

    Eur. J. Med. Res.

    (1997)
  • N. Demaurex et al.

    Intracellular pH regulation during spreading of human neutrophils

    J. Cell Biol.

    (1996)
  • S. Dhein et al.

    The contribution of neutrophils to reperfusion arrhythmias and a possible role for anti-adhesive pharmacological substances

    Cardiovasc. Res.

    (1995)
  • R.L. Engler

    Free radical and granulocyte-mediated injury during myocardial ischemia and reperfusion

    Am. J. Cardiol.

    (1989)
  • Cited by (27)

    • Hypoxia Produces Pro-arrhythmic Late Sodium Current in Cardiac Myocytes by SUMOylation of Na<inf>V</inf>1.5 Channels

      2020, Cell Reports
      Citation Excerpt :

      The hypoxic increase in ILATE we observe with iPS-CMs modeled in the O’Hara-Rudy algorithm are found sufficient to underlie the increase in APD that induces these downstream effects (Figure 5). In addition to hypoxia and ischemia (Saint, 2006; Hammarström and Gage, 2002), cardiac ILATE is reported to increase with oxidative stress (Ward and Giles, 1997), acidosis (Plant et al., 2006), heart failure (Valdivia et al., 2005; Undrovinas et al., 2006), and inflammation (Ward et al., 2006), suggesting a role for SUMOylation in the transduction of these stimuli that merits study. Rapid SUMOylation of NaV1.5 channels on the cardiac plasma membrane in response to hypoxia is reminiscent of our finding that hypoxia mediates SUMOylation of NaV1.2 channels on the surface of central neurons in under 60 s on K38, leading to an increase peak current (without changes in ILATE) due to changes in the voltage dependence of activation, consistent with early, rapid Na+ flux in cerebral ischemia, a prelude to downstream pathology (Plant et al., 2016).

    • Current–Voltage Relationship for Late Na<sup>+</sup> Current in Adult Rat Ventricular Myocytes

      2016, Current Topics in Membranes
      Citation Excerpt :

      Much of our previous work on INa-L is based on our studies of the effects of H2O2 as a prototypical “free radical” in the setting of redox challenge within the compromised myocardium. We have also studied the mechanisms of action of sterile inflammation in some detail (Poon, Ward, Giles, & Kubes, 1999a, 1999b; Ward, Bazzazi, Clark, Nygren, & Giles, 2006), see also Dib-Haji, Black, Cummins, & Waxman, 2002; Maingret, et al., 2008. Our results have shown that one of the primary localized chemical reactions involved in the interaction between emigrated neutrophils and ventricular myocytes also involves the production of H2O2.

    • GATA-4 induces changes in electrophysiological properties of rat mesenchymal stem cells

      2014, Biochimica et Biophysica Acta - General Subjects
      Citation Excerpt :

      Transplantation of MSCGATA-4 can effectively improve cardiac function in the setting of myocardial infarction as well as directly promote their differentiation into CM; however, the impact of GATA-4 overexpression on the electrophysiological properties of MSC remains unclear. Since the transient outward K+ current (Ito), inwardly rectifying K+ current (IK1), TTX-sensitive Na+ current (INa.TTX) and L-type calcium current (ICa.L) are highly expressed in CM and contribute to the electrophysiological actions of CM [17–21], we hypothesized that GATA-4 overexpression would upregulate these functional ion channels (Ito, IK1, INa.TTX and ICa.L) within MSCGATA-4 to promote their differentiation to CM. The present study was designed to investigate the effects of GATA-4 on the electrophysiological properties of MSC as well as uncover potential regulatory mechanisms.

    View all citing articles on Scopus
    View full text