International Journal of Cardiology Lymphocytic myocarditis occurs with myocardial infarction and coincides with increased inflammation, hemorrhage and instability in coronary artery atherosclerotic plaques  Linde Woudstra, P. Stefan Biesbroek, Reindert W. Emmens, Stephane Heymans, Lynda J. Juffermans, Albert C. van Rossum, Hans W.M. Niessen, Paul A.J. Krijnen  International Journal of Cardiology  Volume 232, Pages 53-62 (April 2017) DOI: 10.1016/j.ijcard.2017.01.052 Copyright © 2017 The Authors Terms and Conditions

Fig. 1 Histopathological findings. A) Representative NBT staining of a patients with lymphocytic myocarditis (LM) and a myocardial infarct (MI) of 3–6h old. The infarction area as distinguished by decoloration of NBT staining outlined at the lateral posterior wall. B) The percentage of infarct area of patients with LM+MI and with MI. Data is presented as the mean with standard deviation. C) Example of a CD45 staining with a thrombus (asterisk) coinciding with an atherosclerotic plaque (double asterisk) in the left anterior descending coronary artery of a LM+MI patient. D) Example of an aggregate of CD45+ lymphocytes in the myocardium of a LM patient. NBT: Nitro Blue Tetrazolium. International Journal of Cardiology 2017 232, 53-62DOI: (10.1016/j.ijcard.2017.01.052) Copyright © 2017 The Authors Terms and Conditions

Fig. 2 The grade of stenosis in the coronary artery segments. A) The graph shows the percentage (%) of stenosis in coronary segments of controls, patients with lymphocytic myocarditis (LM), patients with both LM and acute myocardial infarction (LM+MI) and patients with MI (MI). B) The percentage (%) of stenosis of the infarct- and non-infarct related coronary arteries was further subdivided for the LM+MI and MI groups. All the data points are shown combined with a boxplot showing the median and the interquartile range. *p<0.05, **p<0.01, ***p<0.001. International Journal of Cardiology 2017 232, 53-62DOI: (10.1016/j.ijcard.2017.01.052) Copyright © 2017 The Authors Terms and Conditions

Fig. 3 Quantification of macrophages and neutrophils in the coronary artery segments. A) An example of the coronary layers of an anti-CD68 (macrophage) staining. The intima (I), media (M), adventitia (A) and total segment (T) are indicated. B) The number of CD68+ macrophages per mm2 in the total coronary segments and C) subdivided in the different coronary layers of the coronary segments of controls, patients with LM, LM+MI and MI. D) The number of MPO+ neutrophils per mm2 in the total coronary segments and E) subdivided in the different layers of the coronary segments of controls, patients with LM, LM+MI and MI. All the data points are shown combined with a boxplot showing the median and the interquartile. *p<0.05, **p<0.01, ***p<0.001. LM: lymphocytic myocarditis, MI: myocardial infarction. International Journal of Cardiology 2017 232, 53-62DOI: (10.1016/j.ijcard.2017.01.052) Copyright © 2017 The Authors Terms and Conditions

Fig. 4 Quantification of lymphocytes and mast cells in the coronary artery segments. A) The percentage of surface area positive of lymphocytes in the total coronary segments and B) subdivided in the different layers of the coronary segments of controls, patients with LM, LM+MI and MI. C) The number of tryptase+mast cells per mm2 in the total coronary segments of controls, LM patients, LM+MI patients and MI patients and D) subdivided in the different layers of the coronary segments of controls, patients with LM, LM+MI and MI. All the data points are shown combined with as a boxplot showing the median and the interquartile range. *p<0.05, **p<0.01, ***p<0.001. LM: lymphocytic myocarditis, MI: myocardial infarction. International Journal of Cardiology 2017 232, 53-62DOI: (10.1016/j.ijcard.2017.01.052) Copyright © 2017 The Authors Terms and Conditions

Fig. 5 Plaque instability and thrombi in the coronary artery segments. A) Example of a haematoxylin-eosin (HE)-stained stable atherosclerotic plaque of a coronary artery segment. Characteristic is the thick fibrous cap of the intima (I) and absence of inflammatory cells at the luminal endothelial layer of the plaque. B) Example of a HE-stained unstable atherosclerotic plaque of the intima coronary artery segment. Characteristic is the thin fibrotic cap (arrow), the inflammatory cells that reach up to the luminal endothelial layer of the plaque (asterisk) and the ceroid that is present within the plaque (double asterisk). C) The graph shows the percentage (%) of unstable plaques and stable plaques in the of the total atherosclerotic plaques in the coronary segments of controls, patients with LM, LM+MI and MI. D) The graph shows the percentage (%) of coronary segments wherein thrombi were present in controls patients with LM, LM+MI and MI. *p<0.05, **p<0.01, ***p<0.001. LM: lymphocytic myocarditis, MI: myocardial infarction. International Journal of Cardiology 2017 232, 53-62DOI: (10.1016/j.ijcard.2017.01.052) Copyright © 2017 The Authors Terms and Conditions

Fig. 6 Quantification of intraplaque hemorrhage. A–C) Examples of an immunohistological anti-Glut 1 staining identifying extravascular erythrocytes indicative for intraplaque hemorrhage (IPH). In coronary segments wherein no Glut 1+ staining was found IPH was deemed absent (A); Presence of intact Glut 1+ erythrocytes indicates fresh IPH (B); Presence of Glut 1+ residual fragments of erythrocytes indicates old IPH (C). D) Presence of iron based on a histological Perls staining indicates an old IPH. E) The graph shows the percentage (%) of the coronary artery segments in which IPH was present (based on fresh or ongoing IPH) in controls, patients with LM, LM+MI and MI. F) The graph shows the percentage (%) of the coronary artery segments in which either only fresh IPH or ongoing IPH was present in controls, patients with LM, LM+MI and MI. *p<0.05, **p<0.01, ***p<0.001. LM: lymphocytic myocarditis, MI: myocardial infarction. International Journal of Cardiology 2017 232, 53-62DOI: (10.1016/j.ijcard.2017.01.052) Copyright © 2017 The Authors Terms and Conditions