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= Clinically available now. (22.77%)
MSACL 2018 EU : Mezger

MSACL 2018 EU Abstract

Topic: Tissue Imaging

Three Dimensional MALDI-Imaging Mass Spectrometry Mapping of a Calcified Mouse Heart

Stephanie Mezger (Presenter)
M4I, Maastricht University

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Presenter Bio: Born and raised in Eindhoven I also stayed there for my Bachelor’s Biomedical Engineering and Master’s Medical Engineering at the Eindhoven University of Technology. After my graduation I worked at the educational board of the Biomedical Engineering department for two years being involved in assessment policy and quality care among other topics. During this time it became clear to me that I wanted to get back into science, building the bridge between the clinic and more fundamental research. I got this opportunity in the project I am now working on, combining cardiac biomarkers and imaging mass spectrometry at the Maastricht University Medical Center, central diagnostic laboratory and The Maastricht MultiModal Molecular Imaging Institute, division of Imaging Mass Spectrometry.

Authors: S.T.P. Mezger (1,3), A. Jaminon (2), B. Cillero Pastor (1), L. Schurgers (2), A.M.A. Mingels (3), O. Bekers (3), R.M.A. Heeren (1)
(1) The Maastricht MultiModal Molecular Imaging Institute, Division of Imaging Mass Spectrometry, Maastricht University, Maastricht, The Netherlands (2) Department of Biochemistry, Maastricht University, Maastricht, The Netherlands (3) Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands

Short Abstract

Cardiac ischemia may cause myocardial injury followed by cardiac remodelling and dystrophic calcification, this process is not completely understood yet. With this project we aim to establish a (3D) spatial map of lipids, metabolites and peptides using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) on a calcified mouse heart to understand the physiological mechanisms that leads to the process of calcification. Our data reveals specific molecular profiles in the calcified and non-calcified regions, suggesting a switch in the metabolic pathways that occur with remodelling of the heart in the process of calcification. Complementary protein and peptide analysis will help to understand the underlying biological mechanisms related to myocardial injury.

Long Abstract

Introduction

Cardiovascular pathologies are one of the main causes of death worldwide, in particular ischemic heart disease and stroke [1]. Ischemia due to an occlusion in the coronary artery may cause myocardial injury, followed by cardiac remodelling [2, 3]. Other events such as dystrophic calcification can occur in response to inflammation and necrosis [4]. Calcification is an important marker of underlying pathologies, however, the process is not completely understood yet.

The goal of this study is to establish a (3D) spatial map of lipids, metabolites, and peptides using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to understand the physiological mechanisms that leads to calcification of the heart. To gain insight into the molecular level of cardiac calcification we used a mouse model that develops age-related dystrophic cardiac calcinosis [5]. Using MALDI-IMS both molecular and spatial information were gathered and combined in a 2D and 3D model in order to define specific regions of the heart based on their molecular profile.

Methods

Animal model. A DBA/2Nchr mouse used for his study was on a normal diet (water and food ad libitum) for 12 weeks, followed by a high fat diet for another 12 weeks. The mouse was sacrificed at 24 weeks under anaesthesia and perfused with ice-cold calcium free PBS before organs were removed and stored for further analyses. The hearts were immediately snap frozen in liquid nitrogen and stored at -80°C.

Tissue sample. The heart with severe calcification, based on macroscopic observation, was cut at -20°C into 9 µm thick transverse sections, from the heart’s apex to base. The sections were divided over 4 categories (lipids, metabolites, peptides/proteins and immunohistochemistry) and mounted onto ITO-coated conductive slides (Bruker Daltonik GmbH, Bremen, Germany). The slides were stored at -80°C.

MALDI Imaging Mass Spectrometry. Matrix coating was done using the SunCollect automated pneumatic sprayer (SunChrom, Friedrichsdorf, Germany). In brief, for lipid analysis norharmane matrix (7 mg/ml in 2:1 chloroform:methanol) was deposited in a series of 15 layers (the initial layer was sprayed at 10 µL/min, then stepped up from 20 µL/min to 30 µL/min, and subsequent layers were sprayed at 40 µL/min). For metabolite analysis 9-aminoacridine matrix (7 mg/ml in 70% ethanol) was deposited in a series of 11 layers. MALDI-IMS data were acquired on a Rapiflex Tissue Typer MALDI-TOF mass spectrometer (Bruker Daltonik) operating in reflectron mode. For lipids negative and positive polarities (mass range 400-2000) were used and for metabolites negative polarity (mass range 40-1600) was used. Data acquisition was controlled by flexImaging 4.1 software (Bruker Daltonik) with a raster size of 50 µm. 200 laser shots per pixel were summed up to generate each spectrum. To perform dual polarity experiments an offset of 25 µm in y-direction was used.

Data processing. Total ion count (TIC) was used for spectral normalization. Principal component analysis was applied to look for intra-tissue molecular heterogeneity. For molecular identification tandem MS was performed in combination with LIPID MAPS and the Human Metabolome Database search engines.

3D MSI. Individual 2D MALDI-IMS serial sections were merged into a 3D data set using SCiLS software. Co-registration with microscopy optical images was performed after Alizarin Red staining.

Results

MALDI-IMS results revealed heterogeneous distribution of lipids and metabolites in a calcified mouse heart. Region specific molecular profiles were generated after co-registration of MSI data with the stained slides. In particular, a differential distribution was found for lysolipids, phosphocholines, cardiolipins, and phosphatidylinositol’s in the calcified and non-calcified regions. Co-localization with phosphatidylinositol-rich regions was found for some metabolites related to the energy metabolism such as ATP, ADP, and AMP (m/z 505.9, 426.0 and 346.0, respectively), but also for fatty acids such as arachidonic acid (m/z 303.2). Other metabolites like nicotinamide adenine dinucleotide and UDP-glucose (m/z 540.0 and 565.0, respectively) were more present in regions with different grades of calcification. The 3D MSI reconstructed dataset displayed gradual changes in the molecular profiles from calcified to non-affected tissues.

Conclusions & Discussion

2D and 3D MALDI-IMS revealed specific molecular profiles that can be used to differentiate calcified, affected and non-affected myocardial tissue, suggesting a switch in metabolic pathways that occur with remodelling of the heart in the process of calcification. Changes in oxidative, substrate- and energy metabolism have been previously described in heart failure and myocarditis and occur in parallel with changes in protein and enzyme activity [3, 6, 7]. Further analysis at the protein and peptide level will generate complementary data that will help to understand the underlying biological mechanisms related to myocardial injury.


References & Acknowledgements:

1. World Health Organization. The top 10 causes of death. 2017 [cited 2018 18 May]; Available from: http://www.who.int/en/news-room/fact-sheets/detail/the-top-10-causes-of-death.

2. Roffi, M., et al., 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: Task Force for the Management of Acute Coronary Syndromes in Patients Presenting without Persistent ST-Segment Elevation of the European Society of Cardiology (ESC). Eur Heart J, 2016. 37(3): p. 267-315.

3. Heusch, G., et al., Cardiovascular remodelling in coronary artery disease and heart failure. Lancet, 2014. 383(9932): p. 1933-43.

4. Nance, J.W., Jr., et al., Myocardial calcifications: pathophysiology, etiologies, differential diagnoses, and imaging findings. J Cardiovasc Comput Tomogr, 2015. 9(1): p. 58-67.

5. van den Broek, F.A. and A.C. Beynen, The influence of dietary phosphorus and magnesium concentrations on the calcium content of heart and kidneys of DBA/2 and NMRI mice. Lab Anim, 1998. 32(4): p. 483-91.

6. Doenst, T., T.D. Nguyen, and E.D. Abel, Cardiac metabolism in heart failure: implications beyond ATP production. Circ Res, 2013. 113(6): p. 709-24.

7. Remels, A.H.V., et al., NF-kappaB-mediated metabolic remodelling in the inflamed heart in acute viral myocarditis. Biochim Biophys Acta, 2018.


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