recognised to coexist with several other cardiac conditions, notjust LVNC. We describe an innovative approach that utilises fractalalgorithms and high-resolution episcopic microscopy (HREM) tostudy the developmental timing of myocardial trabeculation inmouse and we validate it using a recently described LVNC mousemodel (NOTCH pathway regulator Mib1 mutant).Methods HREM (2–3 μm resolution) analysis was performedprospectively on 123 embryonic mouse hearts consisting of wild-type (WT) NIMR:Parkes, WT C57BL/6 and Mib1flox; cTnT-cremutant and WT littermates. HREM permits the 2D/3D imagingof tissue samples as they are physically sectioned. Datasetsunderwent fractal analysis using a box-counting approach (Figure1).Results LV trabecular complexity showed a significant dropbetween E14.5 and E18.5 (Figure 2). Across all embryonicstages, the apical half of the LV retained the highest fractaldimensions (FD) when compared to the base. By E18.5 the myo-cardium was almost fully compacted registering the lowest FD.For the first time, we demonstrate that strain-specific differencesin LV trabecular patterning exist in mouse because NIMR:Parkescompacts earlier than C57BL/6 (Figure 3). Reslicing experiments(Figure 4) and separate validation tests on Mib1 mutants andWT littermates (Fig.5) confirmed how the proposed methodol-ogy is a reliable and effective tool for the detection of mutagene-sis-related differences in trabeculation.Conclusion Reported here is a method in which sequential, 2Dsections of mouse embryo hearts may be analysed using a fractalalgorithm to calculate ventricular trabecular complexity – a tech-nique so sensitive, that small inter-strain differences in somito-genesis are detectable in mouse pups.

Precise knowledge of the trabecular architecture as it presentsitself in WT, is a prerequisite for the correct identification ofpathological trabecular phenotypes in mouse models of cardiacdisease, explaining the need for a quantitative fractal atlas of tra-becular development.

Fractal mathematics in combination with HREM has thepotential to answer to many developmental biology questions inthe heart, with future applicability to other organ systems and toother species.

C DEVELOPMENT OF WHOLE BODY AND INTRAVASCULARNEAR-INFRARED OPTICAL MOLECULAR IMAGING OFMARKERS OF PLAQUE VULNERABLITY INATHEROSCLEROSIS

1Ramzi Y Khamis, 2Kevin J Woollard, 1Gareth D Hyde, 1Joseph J Boyle, 3Colin Bicknell,4Tetsuya Hara, 4Adam Mauskapf, 5David W Granger, 6Jason L Johnson,4Vasilis Ntziachristos, 2Paul M Matthews, 4Farouc A Jaffer, 1Dorian O Haskard. 1VascularSciences Section, National Hearth and Lung Institute, Imperial College London;2Department of Medicine, Imperial College London; 3Department of Surgery and Cancer,Imperial College London; 4Cardiovascular Research Center and Cardiology Division,Massachusetts General Hospital, Harvard Medical School; 5Biopharm R&D,GlaxoSmithKline, Stevenage, UK; 6Bristol Heart Institute, University of Bristol, Bristol, UK

10.1136/heartjnl-2014-306118.231

Background We aimed to study oxidised low density lipoprotein(oxLDL) as a near infrared fluorescence (NIRF) molecular imag-ing target, using a monoclonal anti-oxLDL autoantibody (LO1)in mouse and rabbit atherosclerosis models.Methods LO1 and an IgG3k control antibody were labelledwith a NIRF dye to give LO1–750 and IgG3–750. Targeting toatherosclerosis in mice was compared to a reporter of matrix-

metalloproteinase activity (MMPSense FAST) by FluorescenceMolecular Tomography (FMT) combined with micro-CT. Uptakeby rabbit aorta was detected with a custom-built intra-arterialNIRF catheter, using IVUS for morphological assessment. Wefurther developed LO1 into a molecularly expressed humanisedFab construct with a cysteine-tag (LO1-Fab-Cys).Results Injection of LO1–750 into high fat (HF) fed Ldlr-/- athe-rosclerotic mice led to specific focal localization within a regionof interest enclosing the aortic arch and its branches. Ex vivostudies confirmed LO1–750 localisation to subendothelium andpartially to macrophages within atherosclerotic lesions. BothLO1–750 and the MMP reporter quantified significant increasesin arterial targeting related to genotype, age, and duration of HFdiet. Following HF diet withdrawal, MMPSense FAST targetingwas affected more dramatically, exhibiting a lower intermediatesignal than that of LO1–750. In the rabbit, LO1–750 localizationwas identified in aortic lesions with the intra-arterial catheter.The LO1-Fab-Cys construct localised to atherosclerosis in themouse model in a similar fashion to the parent antibody.Conclusion We demonstrated the utility of LO1 and derivativemolecularly-expressed constructs, either alone or in combinationwith other reporters as multi-modality optical imaging agents forthe evaluation of atherosclerosis.

D MITOCHONDRIAL DNA DAMAGE CAN PROMOTEATHEROSCLEROSIS INDEPENDENTLY OF REACTIVEOXYGEN SPECIES AND CORRELATES WITH HIGHER RISKPLAQUES IN HUMANS

Emma Yu. Division of Cardiovascular Medicine, University of Cambridge, Addenbrooke’sCentre for Clinical Investigation, Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK

10.1136/heartjnl-2014-306118.232

Introduction Mitochondrial DNA (mtDNA) damage occurs inboth the vessel wall and in circulating cells in human athero-sclerosis. However, whether mtDNA damage promotes athe-rogenesis or is a consequence of tissue damage is unknown.We assessed the hypothesis that mtDNA damage is present,and can directly promote atherosclerosis and affect plaquecomposition.Methods To assess whether mtDNA damage may contribute toatherogenesis we examined apolipoprotein E null mice (ApoE-/-).We characterised the development of atherosclerotic plaques,and concomitantly assessed for mtDNA damage and mitochon-drial dysfunction.

We then studied ApoE-/- mice, also deficient for mtDNA poly-merase γ proof reading activity (polG-/-/ApoE-/-), to determinewhether mtDNA defects directly promote atherosclerosis. Themice were assessed for levels of atherosclerosis, mtDNA damageand mitochondrial respiratory function. We characterised pheno-typic changes in vascular smooth muscle cells (VSMCs) andmonocytes.

We also examined the association between mtDNA damageand human disease. We used qPCR to quantify the levels ofmtDNA damage in human plaques and normal aortic sam-ples. Furthermore, we examined whether leukocyte mtDNAdamage correlates with atherosclerosis extent, or plaquevulnerability.Results MtDNA damage occurred early in the vessel wall inApoE-/- mice, before significant atherosclerosis developed.MtDNA defects were also identified in circulating monocytesand liver, and were associated with reduced respiratory com-plex activity. PolG-/-/ApoE-/- mice showed extensive mtDNA

Young Researcher Workers' Prize 2014

A128 Heart 2014;100(Suppl 3):A1–A138

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doi: 10.1136/heartjnl-2014-306118.231 2014 100: A128Heart

 Ramzi Y Khamis, Kevin J Woollard, Gareth D Hyde, et al. Plaque Vulnerablity in Atherosclerosis

ofNear-infrared Optical Molecular Imaging of Markers Development of Whole Body and Intravascular C

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