Abstract
Background: The proinflammatory cytokine Interleukin-1β (IL-1β), which increases in the heart post myocardial infarction (MI), has been shown to cause loss of Connexin43 (Cx43) function, an event known to underlie formation of the arrhythmogenic substrate. Omega 3 Fatty acids exhibit antiarrhythmic properties and impact IL-1β signaling. We hypothesize that Omega-3 fatty acids prevent arrhythmias in part, by inhibiting IL-1β signaling thus maintaining functional Cx43 channels. Methods: Rat neonatal myocytes or Madin-Darby Canine Kidney Epithelial (MDCK) cells grown in media in the absence (Ctr) or presence of 30 μM docosahexaenoic acid (DHA, an Omega-3 Fatty acid) were treated with 0.1 μM activated IL-1β. We determined Cx43 channel function using a dye spread assay. Western blot and immunostaining were used to examine Cx43 levels/localization and downstream effectors of IL-1β. In addition we used a murine model of MI for 24 h to determine the impact of an Omega-3 fatty acid enriched diet on Cx43 levels/localization post MI. Results: IL-1β significantly inhibited Cx43 function in Ctr cells (200.9 ± 17.7 μm [Ctr] vs. 112.8 ± 14.9 μm [0.1 uM IL-1β], p<0.05). However, DHA-treated cells remained highly coupled in the presence of IL-1β [167.9 ± 21.9 μm [DHA] vs. 164.4 ± 22.3 μm [DHA + 0.1 uM IL-1β], p<0.05, n = 4]. Additionally, western blot showed that IL-1β treatment caused a 38.5% downregulation of Cx43 [1.00 au [Ctr] vs. 0.615 au (0.1 μM IL-1β) which was completely abolished in DHA-treated cells (0.935 au [DHA] vs. 1.02 au [DHA + 0.1 μM IL-1β), p < 0.05, n = 3]. Examination of the downstream modulator of IL-1β, NFκβ showed that while hypoxia caused translocation of NFκβ to the nucleus, this was inhibited by DHA. Additionally we found that a diet enriched in Omega-3 Fatty acids inhibited lateralization of Cx43 in the post-MI murine heart as well as limited activation of fibroblasts which would lead to decreased fibrosis overall. Conclusions: Omega 3 Fatty acid treatment inhibited IL-1β-stimulated loss of Cx43 protein, and more importantly, inhibited loss of Cx43 function by inhibiting translocation of NFκβ. In the intact heart a diet enriched in Omega 3 Fatty Acids limited loss of Cx43 at the intercalated disk in the heart following MI. These data suggest that one of cardio-protective mechanisms by which Omega 3 Fatty acids work includes prevention of the pro-arrhythmic loss of Cx43 post MI and the attenuation of cardiac fibrosis after injury.
Highlights
Gap junctions provide direct electrical continuity between myocytes in the functioning myocardium
Being as NFκβ alters the response of myocytes to the inflammatory cytokine Interleukin 1β, which is upregulated following myocardial infarction (MI) (Abbate et al, 2010), and we have shown that IL-1β affects the gap junction protein Connexin43 (Cx43) in both the nervous system (Duffy et al, 2000) in the injured heart (Baum et al, 2012), we hypothesized that one of the mechanisms for the anti-arrhythmic effects of a diet containing w3 fatty acids is by regulating Cx43 containing gap junctions following myocardial injury
Inflammatory processes in the heart lead to loss of Cx43 therapeutically targeting inflammation may decrease this loss and limit formation of arrhythmias
Summary
Gap junctions provide direct electrical continuity between myocytes in the functioning myocardium. In addition we used a murine model of MI for 24 h to determine the impact of an Omega-3 fatty acid enriched diet on Cx43 levels/localization post MI. We found that a diet enriched in Omega-3 Fatty acids inhibited lateralization of Cx43 in the post-MI murine heart as well as limited activation of fibroblasts which would lead to decreased fibrosis overall. In the intact heart a diet enriched in Omega 3 Fatty Acids limited loss of Cx43 at the intercalated disk in the heart following MI These data suggest that one of cardio-protective mechanisms by which Omega 3 Fatty acids work includes prevention of the pro-arrhythmic loss of Cx43 post MI and the attenuation of cardiac fibrosis after injury
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