Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation.

Fibrosis following myocardial infarction is associated with increases in arrhythmias and sudden cardiac death. Initial steps in the development of fibrosis are not clear; however, it is likely that cardiac fibroblasts play an important role. In immune cells, ATP release from pannexin 1 (Panx1) channels acts as a paracrine signal initiating activation of innate immunity. ATP has been shown in noncardiac systems to initiate fibroblast activation. Therefore, we propose that ATP release through Panx1 channels and subsequent fibroblast activation in the heart drives the development of fibrosis in the heart following myocardial infarction. We identified for the first time that Panx1 is localized within sarcolemmal membranes of canine cardiac myocytes where it directly interacts with the postsynaptic density 95/Drosophila disk large/zonula occludens-1-containing scaffolding protein synapse-associated protein 97 via its carboxyl terminal domain (amino acids 300-357). Induced ischemia rapidly increased glycosylation of Panx1, resulting in increased trafficking to the plasma membrane as well as increased interaction with synapse-associated protein 97. Cellular stress enhanced ATP release from myocyte Panx1 channels, which, in turn, causes fibroblast transformation to the activated myofibroblast phenotype via activation of the MAPK and p53 pathways, both of which are involved in the development of cardiac fibrosis. ATP release through Panx1 channels in cardiac myocytes during ischemia may be an early paracrine event leading to profibrotic responses to ischemic cardiac injury.

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

BACKGROUND: The proinflammatory cytokine Interleukin-1ß (IL-1ß), 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-1ß signaling. We hypothesize that Omega-3 fatty acids prevent arrhythmias in part, by inhibiting IL-1ß 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-1ß. 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-1ß. 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-1ß significantly inhibited Cx43 function in Ctr cells (200.9 ± 17.7 µm [Ctr] vs. 112.8 ± 14.9 µm [0.1 uM IL-1ß], p<0.05). However, DHA-treated cells remained highly coupled in the presence of IL-1ß [167.9 ± 21.9 µm [DHA] vs. 164.4 ± 22.3 µm [DHA + 0.1 uM IL-1ß], p<0.05, n = 4]. Additionally, western blot showed that IL-1ß treatment caused a 38.5% downregulation of Cx43 [1.00 au [Ctr] vs. 0.615 au (0.1 µM IL-1ß) which was completely abolished in DHA-treated cells (0.935 au [DHA] vs. 1.02 au [DHA + 0.1 µM IL-1ß), p < 0.05, n = 3]. Examination of the downstream modulator of IL-1ß, NFκß showed that while hypoxia caused translocation of NFκß 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-1ß-stimulated loss of Cx43 protein, and more importantly, inhibited loss of Cx43 function by inhibiting translocation of NFκß. 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.

Myofibroblasts cause heterogeneous Cx43 reduction and are unlikely to be coupled to myocytes in the healing canine infarct.

Following myocardial infarction (MI) inflammatory responses transform cardiac fibroblasts to myofibroblasts, which in vitro studies show form heterocellular gap junctions with cardiac myocytes via Connexin43 (Cx43). The ability to form heterocellular junctions in the intact heart and the impact of these junctions on propagation is unclear. We used a canine model of MI and characterized the distribution and quantity of myofibroblasts in surviving epicardial cells [epicardial border zone (EBZ)]. We found a significant increase in myofibroblasts within the EBZ and no gap junction plaques between myofibroblasts and myocytes. Because myofibroblasts produce IL-1ß, which downregulates Cx43, we asked whether myofibroblast proliferation causes loss of Cx43 near myofibroblast clusters. In vitro studies showed that IL-1ß caused loss of Cx43 and reduced coupling. Western blot showed a significant increase of IL-1ß in the EBZ, and immunohistochemistry showed a loss of Cx43 in regions of myofibroblasts in the intact heart. Additionally, dye studies in intact heart showed no coupling between myocytes and myofibroblasts. To quantify the effect of myofibroblasts on propagation we used a two-dimensional subcellular computer model of the EBZ, which showed that heterogeneities in myofibroblast density lead to conduction abnormalities. In conclusion, an increase of myofibroblasts in the infarcted heart causes heterogeneous Cx43 levels, possibly as a result of the release of IL-1ß and decreased cell-cell communication, which leads to conduction abnormalities following MI.