Omega 3 Fatty Acid Inhibition of Inflammatory Cytokine-Mediated Connexin43 Regulation in the Heart

Abstract

Background: The proinflammatory cytokine $Interleukin-1\beta$ $(IL-1\beta)$, 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 $IL-1\beta$ signaling. We hypothesize that Omega-3 fatty acids prevent arrhythmias in part, by inhibiting $IL-1\beta$ 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 $30 \mu M$ docosahexaenoic acid (DHA, an Omega-3 Fatty acid) were treated with $0.1 \mu M$ activated $IL-1\beta$. 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 $IL-1\beta$. 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: $IL-1\beta$ significantly inhibited Cx43 function in Ctr cells $(200.9 \pm 17.7 \mu m [Ctr] vs. 112.8 \pm 14.9 \mu m [0.1 \mu M IL-1\beta], p<0.05)$. However, DHA-treated cells remained highly coupled in the presence of $IL-1\beta$ $[167.9 \pm 21.9 \mu m [DHA] vs. 164.4 \pm 22.3 \mu m [DHA + 0.1 \ muM IL-1\beta], p<0.05, n = 4]$. Additionally, western blot showed that $IL-1\beta$ treatment caused a 38.5% downregulation of Cx43 $[1.00 au [Ctr] vs. 0.615 au (0.1 \mu M IL-1\beta)$ which was completely abolished in DHA-treated cells $(0.935 au [DHA] vs. 1.02 au [DHA + 0.1 \mu M IL-1\beta), p < 0.05, n = 3]$. Examination of the downstream modulator of $IL-1\beta$, $NF\kappa \beta$ showed that while hypoxia caused translocation of $NF\kappa \beta$ 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 $IL-1\beta$-stimulated loss of Cx43 protein, and more importantly, inhibited loss of Cx43 function by inhibiting translocation of $NF\kappa \beta$. 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