Omega 3 fatty acid inhibition of inflammatory cytokine-mediated Connexin43 regulation in the heart

Jennifer R. Baum, Elena Dolmatova, Alex Tan, Heather S. Duffy

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.