Cardiovascular Pharmacology
Interleukin-6 impairs chronotropic responsiveness to cholinergic stimulation and decreases heart rate variability in mice

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Abstract

Heart rate variability is reduced in several clinical settings associated with systemic inflammation. The underlying mechanism of decreased heart rate variability during systemic inflammation is unknown. It appears that the inflammatory cytokines might play a role, since epidemiologic studies has shown that circulating levels of interleukine-6 (IL-6) correlate significantly with indexes of depressed heart rate variability in various clinical conditions. The present investigation was carried out to study the peripheral and central effects of IL-6 on heart rate dynamic in mice. Adult male BALB/c mice were used in the study. RT-PCR was performed to study the expression of IL-6 receptor in mouse atrial and the results showed that gp130 mRNA was detectable in the atrium. The effect of IL-6 was also studies on chronotropic responsiveness of isolated atria to adrenergic and cholinergic stimulations. Incubation of isolated atria with 10 ng/ml of IL-6 was associated with a significant hypo-responsiveness to cholinergic stimulation (log IC50 of carbacholine changed from − 6.26 ± 0.10 in controls to − 5.59 ± 0.19 following incubation with IL-6, P < 0.05). The chronotropic responsiveness to adrenergic stimulation was identical with or without incubation with IL-6. Intraperitoneal injection of IL-6 (200 ng/mouse) was associated with a significant decrease in heart rate variability parameters (SDNN, SD1, and SD2). While intracerebroventricular injection of IL-6 (50 ng/mouse) had no significant effect on heart rate variability parameters. These data are in line with a peripheral role for IL-6 in the genesis of decreased heart rate variability during systemic inflammation.

Introduction

Heart rate variations reflect the output of the complex control of the heart mediated by the autonomic nervous system (Akselrod et al., 1981, Altimiras, 1999). Over the last three decades, it has been demonstrated that the beat-to-beat variations of heart rate contain information on the activity of the autonomic effectors controlling cardiac output (Altimiras, 1999). Deceased variability or increased regularity of the cardiac rhythm has been reported in different clinical settings associated with systemic inflammation and increased production of inflammatory cytokines, such as sepsis, diabetes mellitus, ischemic heart disease, congestive heart failure, and hepatic failure; in these contexts, it is a negative predictor of survival (Aronson et al., 2001, Ates et al., 2006, González-Clemente et al., 2007, Griffin et al., 2005, Hamaad et al., 2005, Lanza et al., 2006, Mani et al., 2009).

The underlying mechanism of decreased heart rate variability during systemic inflammation is unknown. Theoretical analysis suggests that greater regularity in a complex system could indicate uncoupling of the system's components (Buchman, 2002, Pincus, 1994). Therefore heart rate variability analysis has been used to study the degree of system isolation in cardiovascular regulation during systemic inflammatory response syndrome. For example, Godin et al. (1996) showed that injection of bacterial endotoxin into human volunteers causes mild uncoupling of autonomic regulation that manifest as loss of heart rate variability in the electrocardiogram signal.

Epidemiological studies have shown a significant correlation between circulating levels of IL-6 and indexes of depressed heart rate variability in various clinical conditions (Aronson et al., 2001, Carney et al., 2007, González-Clemente et al., 2007, Janszky et al., 2004, Mani et al., 2009, Marsland et al., 2007, Shinohara et al., 2008, Tateishi et al., 2007, von Känel et al., 2008). Although these reports suggest a role for IL-6 in reduction of heart rate variability during inflammation, a study that shows a clear cause–effect relationship is missing. A significant correlation between IL-6 and loss of heart rate variability in patients with inflammatory condition might be explained by the direct affect of IL-6 on cardiac pacemaker cells or the autonomic regulatory centers. It could also be a secondary phenomenon that occurs during systemic inflammation in parallel to a primary phenomenon which has a direct effect on cardiovascular regulatory system. It is not clear whether or not injection of IL-6 can mimic the effect of systemic inflammation on heart rate variability. Cardiac pacemaker cells or neural regulatory centers (e.g. vasomotor area in brain stem) might act as a target for inflammatory cytokines leading to changes in heart rate variability. This study was aimed to test whether peripheral or intracerebroventricular (i.c.v.) injection of mouse recombinant IL-6 can decrease heart rate variability in mice. Since cardiac pacemaker cells may act as a target for inflammatory cytokines leading to alteration in heart rate dynamics or their responsiveness to neurotransmitters, we also tested the hypothesis that impaired responsiveness of cardiac pacemaker to autonomic transmitters may lead to uncoupling of the cardiovascular regulatory mechanisms during acute IL-6 challenge.

Section snippets

Chemicals

All reagents were purchased from Sigma-Aldrich (Pool, UK) unless stated otherwise.

Animals

Male BALB/c mice (29–31 g) were used in the experiments. Animals were given free access to normal rodent chow and water and kept at standard condition. All animal procedures were in accordance with “Guide for the Care and Use of Laboratory Animals” (NIH US publication No 85-23, revised 1985) recommendations. Six to eight animals were used in each experimental group.

In vitro study: Preparation of isolated atria

Spontaneously beating atria were then isolated to

Expression of gp130 in mouse atria

To examine the expression of IL-6 receptor in mouse atria, RT-PCR was performed on mRNA extracted from mouse atria using specific primers for an IL-6 receptor subunit (gp130). GAPDH was used as housekeeping gene. Mouse liver was also used as positive control for gp130 expression. As shown in Fig. 1, gp130 and GAPDH were detectable in mouse atria with corresponding PCR products of 303 and 348 bp respectively.

In vitro study

Beating rate and its variability was assessed in spontaneously beating isolated atria

Discussion

Many investigators have shown that during systemic inflammation, physiological time signals (e.g. beat-to-beat interval) would lose the fine variability observed in healthy subjects (Godin et al., 1996, Griffin et al., 2005, Lake et al., 2002, Mani et al., 2006b, Mani et al., 2009, Tateishi et al., 2007). Previous studies have shown that indexes of depressed heart rate variability not only correlate with the extent of disease progression, but also appear to predict adverse outcomes in patients

Acknowledgments

This study was supported by grants from Dean of Research, Tarbiat Modares University, Tehran, Iran. The authors would like to thank Professor S. Semnanian and Dr M. Javan for support.

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