An autoantibody profile detects Brugada syndrome and identifies abnormally expressed myocardial proteins
Authors: Dipashree Chatterjee
Sven Dittmann
J Martjin Bos
Danna A Spears
Gonca Suna
Michael J Ackerman
Diptendu Chatterjee
Leonardo Bolognese
Pasquale G Notarstefano
Robert M Hamilton
Kristopher S Cunningham
Meena Fatah
Eric Schulze-Bahr
Firat Duru
Maurizio Pieroni
Flavien Charpentier
Ardan M Saguner
Journal: European Heart Journal (2020)
Discipline: CLINICAL MEDICINE
Keywords: Actin; Autoantibody; Biomarker; Brugada syndrome; Connexin-43
Affiliations: Department of Pediatrics, The Labatt Family Heart Centre and Translational Medicine, The Hospital for Sick Children & Research Institute and the University of Toronto, Room 1725D, 555 University Avenue, Toronto, ON M5G 1X8, Canada, Cardiovascular Department, San Donato Hospital, Via Curtatone 54 - 52100 Arezzo, Italy, L’Institut du Thorax, INSERM, CNRS, UNIV Nantes, 8 quai Moncousu, 44007 Nantes, France, Department of Laboratory Medicine and Pathobiology, University of Toronto, 27 King's College Circle, Toronto, Ontario M5S 1A1, Canada, Department of Medicine, University Health Network—Toronto General Hospital, 200 Elizabeth Street 4NU-492, Toronto, Ontario M5G 2C4, Canada, Department of Psychology, University of Toronto, 27 King's College Circle, Toronto, Ontario M5S 1A1, Canada, Department of Cardiology, University Heart Center Zurich, Rämistrasse 100, 8091 Zürich, Switzerland, Department of Cardiovascular Medicine, Division of Heart Rhythm Services, Windland Smith Rice Genetic Heart Rhythm Clinic, Mayo Clinic, Rochester, MN, USA, Department of Pediatric and Adolescent Medicine, Division of Pediatric Cardiology, Mayo Clinic, Rochester, MN, USA, Department of Molecular Pharmacology and Experimental Therapeutics, Windland Smith Rice Sudden Death Genomics Laboratory, Mayo Clinic, Rochester, MN, USA, Institute for Genetics of Heart Diseases, Department für Kardiologie und Angiologie, Zentrum für Innere Medizin, Universitätsklinikum Münster Albert-Schweitzer-Campus 1, Gebäude D3 48149 Münster, Germany

Abstract:  Aims: Brugada syndrome (BrS) is characterized by a unique electrocardiogram (ECG) pattern and life-threatening arrhythmias. However, the Type 1 Brugada ECG pattern is often transient, and a genetic cause is only identified in &lt;25% of patients. We sought to identify an additional biomarker for this rare condition. As myocardial inflammation may be present in BrS, we evaluated whether myocardial autoantibodies can be detected in these patients. Methods and results: For antibody (Ab) discovery, normal human ventricular myocardial proteins were solubilized and separated by isoelectric focusing (IEF) and molecular weight on two-dimensional (2D) gels and used to discover Abs by plating with sera from patients with BrS and control subjects. Target proteins were identified by mass spectrometry (MS). Brugada syndrome subjects were defined based on a consensus clinical scoring system. We assessed discovery and validation cohorts by 2D gels, western blots, and ELISA. We performed immunohistochemistry on myocardium from BrS subjects (vs. control). All (3/3) 2D gels exposed to sera from BrS patients demonstrated specific Abs to four proteins, confirmed by MS to be α-cardiac actin, α-skeletal actin, keratin, and connexin-43, vs. 0/8 control subjects. All (18/18) BrS subjects from our validation cohorts demonstrated the same Abs, confirmed by western blots, vs. 0/24 additional controls. ELISA optical densities for all Abs were elevated in all BrS subjects compared to controls. In myocardium obtained from BrS subjects, each protein, as well as SCN5A, demonstrated abnormal protein expression in aggregates. Conclusion: A biomarker profile of autoantibodies against four cardiac proteins, namely α-cardiac actin, α-skeletal actin, keratin, and connexin-43, can be identified from sera of BrS patients and is highly sensitive and specific, irrespective of genetic cause for BrS. The four involved proteins, along with the SCN5A-encoded Nav1.5 alpha subunit are expressed abnormally in the myocardium of patients with BrS. 

Retracted: No

PubPeer comments:
 Figure 3 was brought to my attention by a reader. * In Figure 3B, some blot strips appear to look more similar than expected, as marked with boxes of the same color. Note the shape of the bands, and the position and width of the light darker blue striped appearance of the background. Could the authors post the original uncropped blots, please, to take away any concerns? ![file] , 
 Figure 3C also appears to have some bands that look remarkably similar, marked here by me with pink boxes. Could the authors please check? ![file] A false color version appears to agree that the bands marked with pink boxes in the image above have the same background shading. ![file] , 
 Professor Elisabeth M. Bik (via PubPeer) Dear Professor Bik, Thank you for your comments bringing attention to u2018very similar bands and background stainingu2019 in the figures from two recent papers: 1.tChatterjee D, Fatah M, Akdis D, Spears DA, Koopmann TT, Mittal K, et al. An autoantibody identifies arrhythmogenic right ventricular cardiomyopathy and participates in its pathogenesis. Eur Heart J. 2018;39(44):3932-44. 2.tChatterjee D, Pieroni M, Fatah M, Charpentier F, Cunningham KS, Spears DA, et al. An autoantibody profile detects Brugada syndrome and identifies abnormally expressed myocardial proteins. Eur Heart J. 2020;41(30):2878-90. I will refer to these as ARVC Biomarker and Brugada Syndrome Biomarker respectively. The main reason for the similarities is that, as is standard for the use of protein Western blots when applied repeatedly for the detection of antibodies in large-scale studies, we reuse the protein blot several times after appropriate stripping of the membrane, to look at several sera sequentially. Thus, the background of some blots including the shape of the band used to identify serum antibodies against a specific protein will look similar, as they represent a membrane that has been stripped and re-probed with another serum. However, the densities of the band staining with different sera remain unique. We employed a routinely used protocol from the research supply company for stripping of Western blots. Most research supply companies (e.g. Abcam, Sigma-Aldrich Thermo-Fisher) provide protocols for this procedure, and we used a protocol from Gene Bio-Application Ltd. (GeBA) for our studies. The use of stripping and re-probing of membranes explains the similarities identified within all figures in both papers, except for those specifically addressed below. Additionally, the identification of all serum antibodies was further reconfirmed by enzyme-linked immuno-sorbent assay (ELISA) of each subjectu2019s sample. As requested, we attach the scanned uncropped original blots for the Brugada Syndrome Biomarker paper Figs. 3B and 3C for which the reader made the comments. (See Re Brugada File Fig 3B and 3C) ARVC Biomarker Figure 3 has additional appearance of similarities due to exactly the process that you surmised (a stain on the scanner). For Figure 3 blots BM 7, BM 16, and BM 24, we have checked the original blots in X-Ray film and there was no such background as observed in the paper. Therefore, the spots we see in those 3 blots came from the scanner used to acquire the image of the exposed films. It appears there was a substance on the scanneru2019s plate resulting in the background spots on the image, whereas the original films which has no background. For all image pages from scans performed on that one particular day, spots were identified in the same scanner position on every page. (See attached series of files labeled u2018Original ARVC blots plus scanner artifact 1 through 4u2019). These files also provide some original uncropped blots as requested. For ARVC Biomarker figure 1, The ladder in each row of the figure was meant to be representative of all ladders performed, as including all ladders in our system would have required 8 additional ladders, interspersed every 3 sets of blots. This is because our gel system has 10 wells and each serum sample needs 3 lanes (Dsc2, Dsg2 and N-Cadherin). For each run, we could prepare only sufficient gels for 3 serum samples and an additional lane for the ladder. If we provided all the ladders in a composed picture of 20-30 samples, we would have had one ladder every 3 samples in our figure, which we felt would have resulted in a confusing and even more cramped figure. Of note, we used the pure recombinant proteins of a known molecular weight to create the gels for the probing of sera, and perhaps should simply have provided an arrow indicating the known molecular weight rather than providing a representative ladder. Please note that we have retained the individual ladders for each set of blots, which show very similar separation. Additionally, the identification of anti-DSG2 antibodies was reconfirmed by enzyme-linked immuno-sorbent assay (ELISA) of each serum sample. Finally, the reviewer identified two blots performed with different proteins that they identified as similar: one of ZH 250 keratin 24 and one of ZH 256 connexin 43. When enlarged, the differences between these are evident. (See attached ZH250keratin-ZH256connexin Figure). Please also note that we have completed many blots using the same gel apparatus, using proteins of similar molecular weight and the same amount of protein loaded per lane. As such, you may expect some bands to look similar at low resolution. For example, the above 2 bands (ZH 250 keratin 24 and one of ZH 256 connexin 43) have a similar bending curving because those 2 samples were run in the extreme end lanes in the gel and this bending pattern can sometimes be observed in samples that are run along in those end lanes as we have seen. Sincerely, Drs. Diptendu Chatterjee and Robert M Hamilton ![file] ![file] ![file] ![file] ![file] ![file] ![file] , 
 Hi Authors. Thanks for your response, which is confusing and unfortunately does not address the issues raised. First, it is very confusing to discuss 2 papers in one thread, so let s focus on the Brugada syndrome paper which is in this thread. Dr. Bik raised problems of apparently too similar images in Figure 3, as follows: Regarding Figure 3B, numerous similarities were denoted between samples, as shown here again: ![file] In response you presented  uncropped original blots for the Brugada Syndrome Biomarker paper , showing the image below (cropped from your posting): ![file] There seems to be some confusion here about what an  uncropped original blot should look like. The images you present are not uncropped - they are chopped up just like the ones in the paper. Uncropped means showing the blot image in its original form, with the lanes in the order in which they were run, and preferably with molecular weight markers and any other landmark features that enable the piece of film to be distinguished from others. In addition, the data presented do not show the blots for all the samples. Figure 3B had 12 samples, whereas the  original you present only has 6 samples. Where did the others go? For example, the yellow boxes in Dr. Bik s analysis show similarity between the a-S-actin lanes for patients ZH169 and ZH240. ZH169 is in the  original you present, but ZH240 is not. Likewise samples 250, 256, 258 and 263 are absent. As such there is no way to assert that the original blots support the claims, because (i) they re not original, and (ii) there are lots of them missing. The same occurs for Figure 3C, the control blots. Problems of similarity were as denoted below with blue lines: ![file] You responded with  uncropped original blots for the Brugada Syndrome Biomarker paper , showing the image below (cropped from your posting): ![file] These are not the same images, and again they are cropped and chopped up with no features to navigate the images. FYI, while perusing this paper I found similar problems with Figure 2, wherein several of the  control blots appear to show identical or similar images (contrast has been enhanced to make the similarities easier to see). ![file] There are also similarities in the supplementary files (supplement Fig. 2C, bottom right 2 panels appear the same). Regarding the two images that are claimed to be  totally different , I would err on the side of  more similar than would be expected by chance. The bands and background for the Keratin and the Connexin blots in sample ZH240 are also very similar, and if not the same image then could have been made by different exposures of the same membrane. If the problem is dirt on the scanner, might I suggest the authors clean their scanner and try scanning in the original uncropped blots again? The paper was only published in 2020, so the originals ought to still be available. ,