An autoantibody identifies arrhythmogenic right ventricular cardiomyopathy and participates in its pathogenesis
Authors: Argelia Medeiros-Domingo
Deniz Akdis
Danna A Spears
Qili Zhao
Jeff S Healey
Michael J Ackerman
Bruce M Cattanach
Diptendu Chatterjee
Jason T Maynes
Kirti Mittal
Yu Sun
Robert M Hamilton
Tamara T Koopmann
Meena Fatah
Firat Duru
Muhammad A Rafiq
Johan Martijn Bos
Ardan M Saguner
Corinna Brunckhorst
Journal: European Heart Journal (2018)
Discipline: CLINICAL MEDICINE
Keywords: Arrhythmogenic right ventricular cardiomyopathy; Desmosomal gene mutations; Anti-desmosome antibodies; Desmoglein-2; Ventricular arrhythmias
Affiliations: The Labatt Family Heart Centre (Department of Pediatrics) and Translational Medicine, The Hospital for Sick Children & Research Institute and the University of Toronto, Room 1725D, 555 University Avenue, Toronto, Ontario, Canada., Zurich ARVC Program, University Heart Centre Zurich Department of Cardiology, R√§mistrasse 100, Zurich, Switzerland., University Health Network, Toronto General Hospital Electrophysiology Department, 200 Elizabeth Street, Toronto, Ontario, Canada., MRC Mammalian Genetics Unit, MRC Harwell Institute, Harwell Science and Innovation Campus, Oxfordshire, UK., University of Toronto Department of Mechanical and Industrial Engineering, Kings College Road, Toronto, Ontario, Canada., Population Health Research Institute and McMaster University Department of Medicine (Division of Cardiology), 237 Barton Street East, Hamilton, Ontario, Canada., Mayo Clinic College of Medicine Department of Cardiovascular Medicine, 200 1st St SW, Rochester, MN, USA., Institute of Biomaterials and Biomedical Engineering, University of Toronto, Rosebrugh Building (RS), 164 College Street, Room 40, Toronto, Ontario, Canada., The Hospital for Sick Children and the University of Toronto Department of Anesthesia and Pain Medicine, 555 University Avenue, Toronto, Ontario, Canada., Bern University Hospital, Department of Cardiology, Freiburgstrasse 18, Bern, Switzerland., Center for Integrative Human Physiology, University of Zurich, Winterthurerstr. 190, Zurich, Switzerland.

Abstract:  Aims: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by right ventricular myocardial replacement and life-threatening ventricular arrhythmias. Desmosomal gene mutations are sometimes identified, but clinical and genetic diagnosis remains challenging. Desmosomal skin disorders can be caused by desmosomal gene mutations or autoantibodies. We sought to determine if anti-desmosome antibodies are present in subjects with ARVC. Methods and results: We evaluated ARVC subjects and controls for antibodies to cardiac desmosomal cadherin proteins. Desmoglein-2 (DSG2), desmocollin-2, and N-cadherin proteins on western blots were exposed to sera, in primary and validation cohorts of subjects and controls, as well as the naturally occurring Boxer dog model of ARVC. We identified anti-DSG2 antibodies in 12/12 and 25/25 definite ARVC cohorts and 7/8 borderline subjects. Antibody was absent in 11/12, faint in 1/12, and absent in 20/20 of two control cohorts. Anti-DSG2 antibodies were present in 10/10 Boxer dogs with ARVC, and absent in 18/18 without. In humans, the level of anti-DSG2 antibodies correlated with the burden of premature ventricular contractions (r = 0.70), and antibodies caused gap junction dysfunction, a common feature of ARVC, in vitro. Anti-DSG2 antibodies were present in ARVC subjects regardless of whether an underlying mutation was identified, or which mutation was present. A disease-specific DSG2 epitope was identified. Conclusion: Anti-DSG2 antibodies are a sensitive and specific biomarker for ARVC. The development of autoimmunity as a result of target-related mutations is unique. Anti-DSG2 antibodies likely explain the cardiac inflammation that is frequently identified in ARVC and may represent a new therapeutic target. 

Retracted: No

PubPeer comments:
 This paper looks like complete garbage of science and height of data fabrication clearly visible. Its a pity that the reviewers of a reputed journal like circulation research did not pay attention to these details. Figure 1C: Molecular weight of GPR75 is 59KDa and the band for a linker compound bound to GPR75 is shown below 55kDa. Perhaps its clearly something else bound to 20-HETE and NOT GPR75. If that was the case you would expect the band to be above 60kDa. Figure 3A: This is the worst immunofluorescence image (looks like a complete photoshop). There is clearly no staining happening with GPR75, such a high background and brightness contrast adjusted to increase the red appear darker Figure 3C,D,EF: All the immunoprecipitation bands for GPR57 are below 55 and so thick, its just too good to be true to extract a GPR75 in such high quantities from HUVEC which is not overexpressing it. The band that usually appears around 50KDa is the IgG BAND that is stained with this high of an intensity when doing IP. Perhaps the authors have mistaken since their band for GPR75 is not GPR75 but is IgG. This mistake is repeated all accross the paper and calls for an immediate re-review retraction Systolic blood pressure measured by tail-cuff is no longer accepted (an experienced cardiovascular researcher will tell you can get whatever blood pressure you want by shifting position of the cuff of mouse s tail) All the significant conclusions are based on WB and IP which is clearly flawed and demands for retraction. The editorial reviewers at Circulation research should immediately retract this fake study to maintain integrity of their journal. , 
 Figure 3 and Figure 7: The bands corresponding to GPR75 in the immunoprecipitation (IP) experiments seem to be non-specific IgG heavy chain bands since IgG will also stain at 50kDa. Moreover, based on molecular weight of GPR75 which is 59kDa its band should be observed much higher than shown. The authors have used HUVECs for the purpose of their studies, these cells are without any overexpression of GPR75 and it raises a question as to why the authors have depicted such a strong staining for GPR75 IP d from only 10ug protein, again raising skepticism that it might be the IgG heavy chain band  , 
 Have contacted the senior and corresponding author of this article Dr. Michael Schwartzman at the Medical College of NY? I retired from Vanderbilt in 2014 and since then I am living in Santiago-Chile and , therefore, I don t have now access to the experimental data from which those figures originated. Thanks Jorge H. Capdevila , 
 #2 Agree with this. These bands most likely appear to be IgG bands presented as GPR75 bands, might be a misunderstanding on the part of authors. In my experience of running IP experiments, if the host for detection antibody and immunoprecipitating antibody is the same, IgG bands are often misinterpreted as antigen bands. In this case, it is very likely since the anti-GPR75 antibody (sc-164538) is a goat polyclonal antibody and the same antibody seems to be have used for both pull down and detection. Most of the major conclusions in this rather remarkable study are based on western blotting and IP experiments, which seem to be confounded. I would suggest the authors to release or reviewers editors to request UNCROPPED images of the blots with an appropriate protein ladder. This would solve confusion for the scientific community and help researchers working in similar areas to better understand the mechanisms of GPR75 signaling. , 
 Figure 5C: SiRNAs have an efficiency of 60-80% but in this case it seems to have 100% efficiency as we do not see a band after SiRNA knockdown. Moreover, image doesn t represent quantitative data of only 70-80% reduction. Is the Actin image from the same blot as for GPR75? It appears that less no protein loaded in the lane represented as GPR75 staining after SiRNA knockown. Again calling for release of uncropped images of blots.  , 
 This publication has been very confusing and conflicting. Compelling evidence suggests that what authors seem to be viewing as GPR75 is in fact some other molecule bound to 20-HETE 20Aphedar but not GPR75. It is surprising for me to see that reviewers from Circulation Research have allowed them to insert cropped images of protein ladder next to the blots. This is unacceptable as per NIH publication guidelines for a number of reasons. One simple and major reason is one could cut and paste the ladder to position along the cropped image at whatever position they would like. , 
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 Hello Everyone Thank you all for your comments and suggestions. All of your concerns were raised by the reviewers during the original submission process which led to the addition of several supplemental figures to validate the antibody and answer other questions, meeting all of their recommendations and guidelines in 2017. The Santa Cruz Biotechnology antibodies were invaluable tools with great potency and specificity that unfortunately, like many antibodies in their portfolio, are no longer available. Unfortunately, this has been a major hurdle and has set back many of our studies and those of others in the field. Thus far our pursuit to validate all of the commercially available GPR75 antibodies has led us to have confidence in the Abcam antibody ab75581. This particular antibody thus far through the use of GPR75 siRNA, in our hands, has been very specific for human samples (endothelial cells) but unfortunately so far it has proven to be subpar across mice tissues (WT vs GPR75 KO mice). We are currently working on a manuscript that will further confirm our previous results studies and shed new light into the mechanisms influence by GPR75 which should be ready in the near future (2021). With regards to the siRNA, the Santa Cruz siRNA is very effective and provides great knockdown. We also recommend the siRNA HSS116879 from Thermo Fisher Scientific which is also very good. If you have any questions and need help troubleshooting anything, please feel free to contact us directly as we have several tools that we would love to share and collaborate with anyone interested in dissecting the role of GPR75. , 
 As per the author response, these are claimed to be part of the supplemental material. However, on taking a closer look at the supplemental materials I fail to find any information on: 1. Validity of the antibody 2. Validity of the SiRNA 3. Details on the dose response curves 4. UNCROPPED COMPLETE IMAGES of blots Thanks Jorge H. Capdevila, Epilobium Brachycarpum and Ugyops Stigmata for commenting and addressing critical scientific skepticism associated with this publication. ,