Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis

Myocarditis is an inflammatory disease of the heart that is most commonly caused by a viral infection of the heart. Infections with cardiotropic viruses result in lymphocytic myocarditis (LM), characterized by infiltration in majority of lymphocytes into the heart, that can be accompanied by cardiomyocyte death, through virulence and/or the ensuing immune response.1 The pathological findings in autopsied hearts of LM patients are commonly subtle, with a patchy lymphocytic infiltrate and most often limited myocyte necrosis,2 and myocardium that has an overall normal morphological appearance. Knowledge regarding whether and how the viable morphologically normal myocardium is affected in LM is limited. We previously observed an increased presence of the stress response protein clusterin in the hearts of acute myocardial infarction patients.3 Herein, clusterin was not only present in necrotic cardiomyocytes but also in viable morphologically normal cardiomyocytes in the noninfarcted myocardium. Clusterin is a highly conserved glycoprotein found in most mammalian tissues and body fluids and has many biological functions, including chaperone-like functions,4 complement inhibition5 and apoptosis regulation.6 Moreover, cardiomyocytes were shown to express clusterin in response to ischemia and hypertrophic stimuli. Notably, the expression of clusterin was shown before in the hearts of mice with myosin-induced autoimmune myocarditis.7 The aim of this study was to analyse the presence of clusterin in the hearts of LM patients with a special focus on the morphologically normal myocardium, and in the heart in time after during Coxsackievirus B3 (CVB3)-induced myocarditis in mice. Left ventricular heart tissue was obtained at autopsy from lymphocytic myocarditis (LM; n = 8) patients and controls (n = 4) without any form of heart disease, and immediately snap frozen and stored in liquid N2. This study was approved by the ethics committee of the VU University Medical Center (VUmc), Amsterdam, and conforms to the principles of the Declaration of Helsinki. The use of the leftover material after the pathological examination has been completed is part of the patient contract in our hospital. For this study mouse heart tissue was used from a previous study.8 In short: male C3H mice (Harlan, Horst, the Netherlands) were divided into two groups: uninfected control mice (n = 10) and mice with CVB3-induced myocarditis (n = 68). On day 0, the CVB3 group was injected intraperitoneally (i.p.) with 1 × 105 plaque-forming units of CVB3 (Nancy strain, ATCC, Manassas, VA), while the control group received saline. CVB3-infected mice were terminated on days 4 (n = 10), 7 (n = 10), 10 (n = 9), 14 (n = 10), 21 (n = 10), 35 (n = 10) and 49 (n = 9). The control mice were terminated on day 14. The hearts were excised, fixed in formalin and then embedded in paraffin. The study was approved by the VU animal ethics and welfare committee and conforms to the Guide for the care and use of laboratory animals published by the US National Institutes of Health. The presence of clusterin, CD45+ cells and C3d (activated complement as a marker of cell death) in human hearts was examined in serially-cut acetone-fixed sections (4 μm). The slides were blocked with Normal Rat Serum (1:50, Dako X0902) for 10 min (for clusterin and CD45) or with H2O2/methanol (for C3d) for 30 min. The slides were then incubated with mouse-anti-human clusterin antibody (1:30, mAb G73), mouse-anti-human CD45 (1:200, Dako M0701) and rabbit-anti-human C3d (1:1000, Dako A0063) for 60 min. For secondary antibodies HRP-conjugated rabbit-anti-mouse immunoglobulins HRP (1:25, Dako P0260; for clusterin and CD45) for 60 min or EnVisionTM+ HRP System anti-rabbit (Dako K4003; for C3d) for 30 min were used, followed by subsequent incubation in DAB, haematoxylin and H2O + NH3. Clusterin and early cell damage (using a histological phosphotungstic acid–haematoxylin stain [PTAH] stain) were examined on 4 μm cross sections. After antigen retrieval by heat activation in citrate buffer (0.01 M, pH 6.0), the slides were incubated with rabbit-anti-mouse clusterin antibody (1:100, Boster PA2233) for 60 min, followed by EnVisionTM+ HRP System anti-rabbit (Dako K4003) for 30 min and visualization by subsequent incubation in AEC single solution (ThermoFisher 001122), haematoxylin and H2O + NH3. Clusterin+ cardiomyocytes, C3d + cardiomyocytes and CD45+ lymphocytes were quantified using light microscopy. In the mouse hearts, only clusterin+ cardiomyocytes outside the myocarditis lesions were counted, while the numbers of CD45+ lymphocytes and lesion size in the mouse hearts were obtained from our previous study in these mice.8 The numbers of positive cells were calculated per mm2 of the tissue surface areas measured on scanned slides using a PathScan Enabler IV slide scanner (Meyer Instruments Inc., Houston, TX, USA) and QuickPHOTO MICRO 3.0 software. Differences between groups were analysed for statistical significance using GraphPad Prisms 7.02 software, using One-way ANOVA with post hoc Tukey's range test. Dunn, Mann–Whitney U or unpaired t-tests were used when appropriate. p values of