{
  "abstract": "Introduction Carbon monoxide (CO) is an air pollutant that people are chronically exposed to in urban environments, however, little is known about its effect during cardiac development. Epidemiological data has observed links between CO exposure and congenital heart defects. Furthermore, recent studies have shown links between CO exposure and ventricular hypertrophy, however little is known about the pathology that causes these phenotypes. The avian model is used to study the in vivo effects of CO exposure due to similar morphology and high genetic conservation. In ovo development also removes maternal confounding factors. To investigate molecular pathways perturbed, we utilised the H9c2 cell line, a rat model of embryonic cardiomyoblast cells. We aimed to use μCT as a non-destructive tool to assess structural morphology, such as lumen volume, which is not possible with traditional methods, alongside molecular techniques to investigate pathophysiological mechanisms. We hypothesise that CO could impact hypoxic and mitochondrial mechanisms that may impact cardiac development, such as preventing the delivery of oxygen to cells and inhibiting mitochondrial function.Methods Fertilised chicken eggs (n=4 to 6 per condition) ( Gallus gallus) were separated into incubation boxes and exposed to a range of CO levels (10, 20, 40, 80, 120ppm). On day 10, hearts were excised and fixed in PFA or snap-frozen for protein extraction. PFA fixed samples were processed for iodine-enhanced μCT imaging or FFPE embedding. Snap-frozen samples were lysed in RIPA. H9c2 cells were exposed to CO for 72 hours prior to cell-based assays or protein extraction. Western blotting (WB) was performed to identify protein levels across groups.Results μCT analysis ( figure 1B) did not detect any significant changes in a range of quantification approaches. We investigated the molecular mechanisms of the HIF1-ET1 hypertrophy axis, where a non-significant downwards linear trend in relative HIF1 levels (figure 2Bi), and a non-significant change in relative ET-1 levels (figure 2Bii) was observed.Discussion μCT is a promising tool for non-destructive imaging of cardiac specimens, but volumetric analysis has limitations, such as defining segments for quantification, which includes compact and trabeculae myocardium, this conflicted with previous histological analysis which observed an increased thickness of the compact myocardium. We also observed no significant structural changes of interest. After μCT-scanning, samples were recovered for FFPE embedding and histology. At the levels studied, CO exposure does not mediate any significant HIF1 change, and hypoxia is not present or does not exceed the threshold to affect HIF1 protein accumulation, however a negative linear trend is observed, and further work is ongoing to look at transcription levels of HIF1 and downstream target VEGFA. WB analysis revealed no statistical change in ET-1 levels.Abstract 386 Figure 1Reconstructed μCT projections to visualise gross heart morphology. A: (i) Coronal and (ii) transverse plane sections segmented into regions of interest. (Yellow-right ventricular lumen; blue-right ventricular wall; green left ventricular lumen; and red-left ventricular wall); B: (i) Left and (ii) right-wall to lumen ratiosAbstract 386 Figure 2Western blot analysis of HIF-1α and ET-1. A: Representative western blot bands for HIF1 and ET1; B: Scatter plots showing the relative protein of (i) HIF1 and (ii) ET1 in cardiac specimens taken from embryos exposed to CO. Samples were snap frozen and normalised against a reference sample used on every blot",
  "authors": [
    {
      "affiliations": [
        "Sheffield Hallam University, Sheffield, United Kingdom"
      ],
      "name": "Joshua Durrans"
    },
    {
      "affiliations": [
        "Manchester Metropolitan University, Manchester, United Kingdom"
      ],
      "name": "Liam Ridge"
    },
    {
      "affiliations": [
        "Sheffield Hallam University, Sheffield, United Kingdom"
      ],
      "name": "Prachi Stafford"
    },
    {
      "affiliations": [
        "Sheffield Hallam University, Sheffield, United Kingdom"
      ],
      "name": "Mari Herigstad"
    }
  ],
  "title": "386 Assessing structural and molecular effects of chronic carbon monoxide exposure on early heart development in an avian model",
  "uid": "a6980a56-8e4d-5055-9073-d17f81f4667a"
}