Cardiac rhythm and pacemaking abnormalities in patients
affected by endemic pemphigus in Colombia may be the
result of deposition of autoantibodies, complement,
fibrinogen, and other molecules
Ana Maria Abreu Velez, MD, PhD, DSc,* Michael S. Howard, MD,*
Jorge Enrique Velazquez-Velez, MD†
From the *Georgia Dermatopathology Associates, Atlanta, Georgia, and †Department of Cardiology,
Hospital General de Medellin, and Clinica CES Medellin, Antioquia, Colombia.
BACKGROUND We previously showed that one-third of patients
affected by endemic pemphigus foliaceus in El Bagre, Colombia
(El Bagre-EPF), display autoreactivity to the heart.
OBJECTIVE The purpose of this study was to investigate rhythm
disturbances with the presence of autoantibodies and correlate
them with ECG changes in these patients.
METHODS We performed a study comparing 30 patients and 30
controls from the endemic area, matched by demographics,
including age, sex, weight, work activities, and comorbidities.
ECG as well as direct and indirect immunofluorescence, immunohistochemistry, and confocal microscopic studies focusing on cardiac
node abnormalities were performed. Autopsies of 7 patients also
were reviewed.
RESULTS The main ECG abnormalities seen in the El Bagre-EPF patients were sinus bradycardia (in one-half), followed by left bundle
branch block, left posterior fascicular block, and left anterior fascicular block compared with the controls. One-third of the patients
Introduction
Endemic forms of pemphigus foliaceus are a unique group of
autoimmune diseases, and the diseases frequently run in families.1–4 These disorders offer an outstanding opportunity to
study interactions of the environment and genetics with the
immune system.1–4 These diseases are characterized by
restriction to relatively well-defined geographic regions of
South and Central America and Tunisia, Africa.1–4 We
previously described a new variant of endemic pemphigus
foliaceus in El Bagre, Colombia (El Bagre-EPF; pemphigus
This work was funded by Georgia Dermatopathology Associates; Mineros SA, Medellin, Colombia; Hospital Nuestra Se~nora del Carmen, El Bagre, Colombia; The Embassy of Japan in Colombia and the El Bagre
Mayoral Office. All authors have reported that they have no relationships
relevant to the contents of this paper to disclose. Address reprint requests
and correspondence: Dr. Ana Maria Abreu Velez, Georgia Dermatopathology Associates, 1534 North Decatur Rd NE, Suite 206, Atlanta, GA 303071000. E-mail address: abreuvelez@yahoo.com.
1547-5271/$-see front matter © 2017 Heart Rhythm Society. All rights reserved.
displayed polyclonal autoantibodies against the sinoatrial and/or
AV nodes and the His bundle correlating with rhythm anomalies
and delays in the cardiac conduction system (P ,.01). The patient
antibodies colocalized with commercial antibodies to desmoplakins
I and II, p0071, armadillo repeat gene deleted in velo-cardio-facial
syndrome (ARVCF), and myocardium-enriched zonula occludens-1associated protein (MYZAP; Progen Biotechnik) (P ,.01).
CONCLUSION One-third of the patients affected by El Bagre-EPF
have rhythm abnormalities that slow the conduction of impulses
in cardiac nodes and the cardiac conduction system. These abnormalities likely occur as a result of deposition of autoantibodies,
complement, and other inflammatory molecules. We show for the
first time that MYZAP is present in cardiac nodes.
KEYWORDS Autoimmune disease; Endemic pemphigus foliaceus;
Rhythm abnormality; Sinus bradycardia
(Heart Rhythm 2018;-:1–7)
All rights reserved.
©
2017 Heart Rhythm Society.
Abreu-Manu). The disease presents in gold and other mining
areas polluted with mercury and other metals and metalloids,
and with severe deforestation.5–9 El Bagre-EPF occurs in
several clinical and immunologic forms, including fruste,
bullous, foliaceus, erythrodermic, pustular, hyperpigmented,
papillomatous, and a generalized form with systemic anomalies. The milder form is localized to the skin. We have
demonstrated a systemic form in about one-third of the patients that affects several organs, including the kidney. It is
characterized by episodic relapses, a tendency toward chronicity, challenging treatment, and a worse prognosis
compared to the localized form.5–11 The systemic form
also affects the cardiovascular system, including its
neurovascular junctions.5–7 We previously showed cardiac
anomalies, including sudden death syndrome and syncope,
and autoantibodies to several parts of the heart in El BagreEPF patients.7 We now aim to continue investigating cardiac
rhythm disturbances in these patients and to correlate them
with the presence of antibodies against cardiac structures.
https://doi.org/10.1016/j.hrthm.2017.12.023
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Methods
We performed a pilot study testing 30 patients affected by El
Bagre-EPF and 30 controls from the endemic area matched
by demographics (age, sex, and weight), work activities,
comorbid acute and/or chronic conditions, exposure to chemicals, socioeconomic status and income, exposure to sun,
mobility and changes in residences, food intake, medications,
and exposure to insect bites. We used a paired case-control
study, correlating the presence of autoantibodies against the
cardiac conduction system vs a lack of immunologic findings
in the controls. The patients were also evaluated epidemiologically and clinically by ECG, skin biopsies (hematoxylin
and eosin [H&E] staining), direct and indirect immunofluorescence (DIF, IIF), immunohistochemistry (IHC), and Airyscan confocal microscopy (CFM) staining. Only patients
who met diagnostic criteria for El Bagre-EPF were included,
specifically: (1) patients who displayed clinical and epidemiologic features described for this disease5,6; (2) patients who
lived in the endemic area; (3) patients whose serum displayed
intercellular staining between epidermal keratinocytes and
the basement membrane zone of the skin by either DIF
or IIF using fluorescein isothiocyanate–conjugated
monoclonal antibodies to human total immunoglobulin (Ig)
G or IgG4, as previously described elsewhere5,6; (4) each
patient whose serum was positive by immunoblotting for
reactivity against Desmoglein 1 (Dsg1), as well as for plakin
molecules as previously described5–9; (5) each patient whose
serum immunoprecipitated a concanavalin A affinity-purified
bovine tryptic 45-kDa fragment of Dsg18; and (6) each patient whose serum yielded a positive result using an enzyme
linked immunosorbent assay when screening for autoantibodies to EPF antigens.9 Written consent was obtained
from all patients, and permission was obtained from the institutional review board at Hospital Nuestra Se~
nora de El Carmen, El Bagre, Colombia. All patient cases and controls
were tested using the same methods and under the same conditions. Positive and negative controls were performed for all
tests. We recorded patient demographics, weight and obesity
measurements, comorbidities, work activities, walking distances to homes and workplaces, and exposure to environmental risk factors. After these measurements, “matched
controls” were selected as a control group. The matched controls were deemed by these criteria to be at similar risk as the
El Bagre-EPF patients for development of the disease. We
also performed H&E and IHC studies for 7 autopsies of
patients who had died affected by EPF. No ECG data were
available for these 7 patients, and they were not part of the
study group of 30 patients.
IIF and DIF
In brief, we dissected the hearts of cows and localized the
sinoatrial and AV nodes, and the His–Purkinje system. We
then used H&E staining and IHC to verify their nature. For
IIF, we incubated 4-mm-thick antigen tissue from each
node and the His–Purkinje system with patient and control
sera. For DIF, we incubated skin tissue from each patient
and/or control with secondary antibodies as previously
described.7–9 We incubated the slides with phosphatebuffered saline (PBS) and 3.5% paraformaldehyde. The
slides were then washed twice with PBS, permeabilized
using PBS with 0.1% Triton X-100, blocked with 1% normal
goat serum, and washed with PBS. We then applied rabbit
anti-human total IgG, IgA, IgM, k and l light chains, and
C1q and C3 antibodies to the slides. We also used antibodies
against fibrinogen and albumin. All of the preceding antibodies were obtained from Dako (Carpinteria, CA). In addition, anti-human IgE antiserum was obtained from Kent
Laboratories (Bellingham, WA) and anti-human IgD antibodies from Southern Biotechnology (Birmingham, AL).
The DIF slides were counterstained with DAPI (Pierce,
Rockford, IL).5–9 Several years ago, Dr. Abreu discovered
new El Bagre-EPF autoantigens to several organs other
than the skin. Because of the complexity of the immune
response, we contacted other experts in the field, including
Dr. Ernest H. Beutner in the United States, Dr. Takashi
Hashimoto in Japan, and Dr. Werner W. Franke, exprofessor at the University of Heidelberg, Germany. All
agreed that the data indicated new autoantigens and that the
disease was unique. A few months later, the primary owner
of Progen Biotechnik (Dr. Franke) illegally commercialized
them without Dr. Abreu’s permission. We thus used antibodies to desmoplakins 1 and 2 (DPI/II; catalog no. 65146,
Progen Biotechnik, Heidelberg, Germany). We used Progen
antibodies to armadillo repeat gene deleted in velo-cardiofacial syndrome (ARVCF) (catalog no. GP155); for its
secondary, we used Alexa Fluor555 goat anti-guinea pig
(ThermoFisher Scientific, Waltham, MA). We also used a
Progen antibody to p0071 (catalog no. 651166) and a Progen
antibody for myocardium-enriched zonula occludens1-associated protein (MYZAP; catalog no. 651169). As a
secondary antibody for the DPI/II, p0071, and MYZAP, we
used Texas red conjugated goat anti-mouse IgG (ThermoFisher). We classified our findings as negative (2), weakly
positive (2/1), positive (1), or strongly positive (111).
We also used an additional antibody to study colocalization
in the heart: rabbit anti-connexin 43 (Sigma Aldrich, St.
Louis, MO); for its secondary, we used Texas red conjugated
goat anti-rabbit IgG.
Colocalization of patient autoantibodies with
commercial antibodies using CFM
Our CFM studies were performed as previously described.7
Standard 20! and 40! objective lenses were used. Each frame
included an area 440 ! 330 mm. Images were obtained using
EZ-1 image analysis software (Nikon, Tokyo, Japan). For
colocalization experiments with serum autoantibodies, we
used the antibodies to DPI-II, ARVCF, p0071, and MYZAP.
IHC
Our studies were performed as previously described.7,10
We tested for mouse anti-human IgG, anti-human C3c,
a-1-antitrypsin, human matrix metalloproteinase-9, human
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Endemic Pemphigus and Autoantibodies to Cardiac Nodes in El Bagre, Columbia
tissue inhibitor of metalloproteinase-1, metallothionein, and
urokinase (all from Dako). We also tested 7 autopsy cardiac
nodes from El Bagre EPF patients who died in the endemic
area.
Airyscan CFM
We used the Zeiss LSM 880, a 3-channel (1GAsP 2PMT)
NLO-ready spectral confocal system with Airyscan (highresolution confocal imaging 170 nm, measured at 130 nm
on delivery). We used 6 visible excitation lines (405, 458,
488, 514, 561, 633 nm) and 6D acquisition (XYZTlP, XZ,
YZ, mosaics) with simultaneous and sequential scanning.
Blood smear testing
Blood smear testing for the Trypanosoma cruzi parasite via
microscopic examination in the cases and controls was
consistently performed.
ECG and blood pressure studies
ECG and blood pressure studies were performed using
routine techniques. ECGs were not available for the 7 autopsied patients because they did not die in the hospital.
Statistical analysis
We used the Fisher exact test to compare 2 nominal variables
(e.g., positivity and negativity of the antibodies). We also
compared the differences when evaluating positivity of the
El Bagre-EPF autoantibodies between patient cases and controls, and patient antibody results vs the commercial antibodies to MYZAP, p0071, DPI/II, and ARVCF. We also
compared comorbidities (hypertension, diabetes, heart failure, and medications, especially those that alter heart rate,
beta-blockers, calcium channel blockers, and antiarrhythmic
drugs). P ,.01 with 98% confidence interval was considered significant. GraphPad QuickCalcs software (GraphPad
Software, La Jolla, CA) was used.
3
All of the El Bagre-EPF patients who displayed autoantibodies to the cardiac nodes had chronic disease. Blood pressure data revealed that 3 El Bagre-EPF patients were poorly
treated for hypertension or had received no treatment; these
patients displayed no rhythm abnormalities. One control
with untreated psoriasis and untreated hypertension also
showed a left anterior fascicular block (see Supplementary
Table 1).
DIF, IIF, and CFM
Our testing demonstrated positive staining with El BagreEPF autoantibodies for either sinoatrial and/or AV nodes of
the His bundle in about one-third of the El Bagre-EPF
patients by different methods, including IIF, CFM, and
IHC (Figures 1 and 2). The presence of autoantibodies using
IIF and respective titers in the study patients vs controls is
shown in Supplementary Table 1. We demonstrated the autoimmune response to be polyclonal as observed by DIF, IIF,
IHC, and CFM, and to involve IgG, IgM, C1q, C3c, fibrinogen, albumin, k and l light chains, and in some cases
IgA, IgD, and IgE. Only 3 controls from the endemic area
were positive: 1 with systemic sclerosis in the His bundle
positive for IgM and C3c at low antibody titers, and 2 with
extensive active psoriasis (P ,.01) (see Supplementary
Table 2). A correlation existed between skin autoantibodies
using DIF and the deposits of antibodies in the cardiac nodes
and the His bundle using IIF. Overall, 16 of 30 patients
affected by El Bagre-EPF demonstrated autoantibodies that
colocalized with the ARVCF, DPI/II, MYZAP, and p0071
obtained from Progen (P ,.01). Furthermore, we observed
that all of the Progen antibodies were present in the cardiac
nodes and the cardiac conduction system, including the His
bundle. All patients and controls expressed MYZAP,
ARVCF, p0071, and DPI/II, indicating that these molecules
are constitutive components of these structures. The controls
showed negative findings by most methods, except for the
three cases we document above.
Results
The most common abnormality found in 53.3% of El BagreEPF patients was sinus bradycardia in 16 of 30 patients
(P ,.01) vs 3 of 30 in the control group. The second most
common rhythm anomaly was left bundle branch block
seen in 4 of 30 El Bagre-EPF patients and in no controls.
Left posterior fascicular block was seen in 2 of 30 El
Bagre-EPF patients and in no controls. Left anterior fascicular block was seen in 1 of 30 El Bagre-EPF patients and 1
of 30 controls (Figure 1 and Supplementary Table 1).
One control patient affected by systemic sclerosis showed
first-degree AV block and left anterior fascicular block. One
El Bagre-EPF patient presented with a premature ventricular
complex, which originated from the right ventricular outflow
tract. One obese female control affected by postinflammatory
hyperpigmentation also showed sinus tachycardia and left
atrial dilation.
In several El Bagre-EPF cases, no symptoms were
detected clinically despite a documented ECG abnormality.
Blood smear testing
Blood smear testing for the Trypanosoma cruzi parasite via
microscopic examination was negative in all the subjects of
the study.
We found positive staining for tissue inhibitor of
metalloproteinase-1 and a-1 antitrypsin in the nodes and cardiac conduction system using IHC in 5 of 7 patient autopsies.
Matrix metalloproteinase-9 staining was positive in the endothelial cells of 3 of 7 El Bagre-EPF patients. The El
Bagre-EPF autopsy patients died at home or at work, and
no ECGs had been performed before the deaths of these patients. Pertinent data obtained after the autopsies are given in
Supplementary Table 3. The 7 patients who had undergone
autopsies were not part of the study group of 30 patients.
Information on autoantibody staining using IIF on study
patients and controls and their respective autoantibodies
using bovine heart tissue antigen substrates is given in
Supplementary Table 1.
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Figure 1 a: ECG of first-degree AV block in an El Bagre-EPF patient. b: ECG of left bundle branch block in an El Bagre-EPF patient. c, d: Confocal microscopic data showing positive staining with El Bagre-EPF autoantibodies labeled with FITC-conjugated anti-human immunoglobulin G (green staining) (111)
colocalizing with a Texas red conjugated commercial antibody to MYZAP (red staining; white arrows) in the AV node. Note in c the peaks of the fluorochromes
(FITC and Texas red) show perfect colocalization, correlating to the peaks (white arrows). EPF 5 endemic pemphigus foliaceus; FITC 5 fluorescein isothiocyanate; MYZAP 5 myocardium-enriched zonula occludens-1-associated protein.
The basic characteristics of some of the cases and controls,
and comorbidities such as diabetes, blood pressure, and ECG
results are compared in Supplementary Table 2.
Information on the 7 El Bagre-EPF patients who died and
their primary autopsy findings is given in Supplementary
Table 3. These patients died at home or at work, and no
ECG data are available for them.
The graphic diagram shows the putative areas where the
autoantibodies may be causing damage and/or rhythm abnormalities.
Discussion
In the current study, we focused on cardiac conduction
system and node abnormalities in patients affected by El
Bagre-EPF (one-half of patients and 3 controls from the
endemic area). The data showed sinus bradycardia in
one-half of the patients compared with the controls.12 El
Bagre-EPF shares many characteristics with Senear-Usher
syndrome (with clinical characteristics of both lupus and
pemphigus).5,6 We hypothesized that the observed rhythm
disturbances found in our study could be correlated with
deposits of polyclonal antibodies and fibrinogen/
complement/albumin in the sinoatrial and AV nodes, His
bundle, and Purkinje system. We previously reported that
one-third of El Bagre-EPF patients have cardiac autoantibodies (mainly to cell junctions) in several areas, which
may lead to syncope and sudden death.7 In the current study,
we focused on cardiac rhythm abnormalities.
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Abreu Velez et al
Figure 2 El Bagre-EPF autoantibodies colocalizing with ARVCF, MYZAP, DPI/II, and p0071 in a sinoatrial node. a: Example of negative staining of the
sinoatrial node using the serum of a control patient on bovine heart. Note positive staining against the cell junctions (red staining; white arrows) of the antibody
against MYZAP (111) (magnification 1000!). b: Immunohistochemistry using an El Bagre-EPF patient autopsy sinoatrial node demonstrating positive
staining using anti-human complement/C3c (brown staining; black arrow) (111) in the node. c: Confocal microscopy of bovine heart antigen showing positive
staining for MYZAP (111) (red staining; white arrows). d: El Bagre-EPF patient autoantibodies labeled with FITC-conjugated anti-human immunoglobulin
M (IgM) antibodies (green staining; white arrows). f: Nucleus of the cells shown in d is stained with DAPI in blue (nuclei of the cardiac nodes are shown by
white arrow). e: Note colocalization of MYZAP and El Bagre-EPF IgM antibodies (combining c and d) (white arrows) (magnification 1000!). DPI/II 5 desmoplakins 1 and 2; EPF 5 endemic pemphigus foliaceus; FITC 5 fluorescein isothiocyanate; MYZAP 5 myocardium-enriched zonula occludens-1-associated
protein.
We also showed that most of the patients have
polyclonal autoantibodies/fibrinogen/complement/albumin
directed to cardiac nodes and the His bundle detected by
multiple immunologic methods in comparison to controls.
No specific demographic or epidemiologic risk factors
were associated with these findings, including obesity,
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6
hypertension, or intake of antiarrhythmic medications. The
Public Health System in Colombia primarily uses antidiuretics as first-line treatment of hypertension.
We speculate that the pathologic deposits of autoantibodies and inflammatory molecules attacking cell junctions
(as demonstrated by colocalization with MYZAP, ARVCF,
DPI/II, and p007) may cause cell separation and alterations
in protein structure within cell junctions of the conduction
system and the neurovascular structures feeding the cardiac
nodes. These structural changes could in turn delay cardiac
impulses, causing sinus bradycardia and other rhythm alterations. These deposits could also lead to phagocytosis and alterations in the sinoatrial and AV nodes, as well as in the His
bundle. Desmoplakins represent components of the bundle of
His–Purkinje cell junctions. These structures are involved in
cell-to-cell communication, so the desmoplakins likely
would be altered. These cell junctions allow rapid conduction
of impulses, and assuming there are autoantigens located in
the cardiac nodal conduction system and the Purkinje system
and His bundle, impulses would likely be delayed.13 The
vascular cell junctions supplying the cardiac nodes also
have autoantibodies in their cells junctions, likely contributing to the sinus bradycardia.14
We also speculate that direct damage to the autonomic
nervous system associated with the vessels, specifically neural receptors in the vascular adventitia, cardiac atria and ventricles, and sinoatrial and AV nodes, may be contributing to
these alterations. We previously showed autoantibodies to
the neurovascular system in the majority of El Bagre-EPF
patients.13,14
We also have reported the presence of mercuric sulfides/
selenides, and other metals and metalloids using autometallography and electron microscopic studies in cell junctions
where El Bagre-EPF autoantigens are present. These autoantigen molecules include Dsg1, DPI/II, and others.15
Endemic forms of pemphigus foliaceus are a unique group
of autoimmune diseases that frequently present in families,16 indicating both a genetic element and environmental
trigger factors to its endemic nature.5,6,15 The people in El
Bagre use mercury, sulfur, and cyanide to facilitate gold
ore extraction, and these metals can compete with
physiologic ions such as calcium and potassium, or they
can cause conformational changes in molecules of the
cardiac conduction system and thus create antigenicity.15
Methylmercury may also (1) affect the release of neurotransmitters from presynaptic nerve terminals; (2) alter calcium intracellularly and alter the permeability of the plasma
membrane; and (3) alter selected ion channels, shifting
action potentials and other electrical signals by gating the
flow of ions across the cell membranes of the cardiac conduction system.17
Another putative pathogenic mechanism for causing the
rhythm alterations is modification of cell signaling, as DPI/II,
p0071, ARVCF, and MYZAP all are present in cell junctions.18,19 MYZAP plays a role in cellular signaling via
Rho-related GTP-binding proteins and activation of transcription factor SRF.20 p0071 regulates Rho signaling during
cytokinesis and is essential for cell division.20 In contrast, inhibition of Rho family protein actions by overexpression of
Rho GDP dissociation inhibitor resulted in AV block with
atrial enlargement and ventricular hypertrophy.21,22
DPI/II, p0071, ARVCF, and MYZAP are present in cardiac myocytes, blood vessels, and nerves and their cell junctions.17–24 The cardiac conduction system includes
desmosomes, gap junctions, fascia adherens junctions, and
composite junctions (area composita).17–24 The sinoatrial
pacemaker complex has desmosomes (desmoplakin and
plakoglobin) and gap junctions (connexins 30 and 45).18–25
The AV node has desmosomes (desmoplakin), adherens
junctions, N-cadherin, and gap junctions (connexins 40 and
45).17–24 The His bundle/penetrating branches have
desmosomes
(desmoplakins),
adherens
junctions
(N-cadherin), and gap junctions (connexins 40 and 45).
The Purkinje network has desmosomes (desmoplakins,
Dsg2), adherens junctions (N-cadherin), composite
junctions (desmoplakins, Dsg2, b- and N-cadherin), and
gap junctions (connexins 40 and 54).18–25 Therefore, El
Bagre-EPF autoantibodies may be altering heart rhythm in
these patients.
The second most common cardiac rhythm anomaly seen
in the El Bagre-EPF patients was left bundle branch block.
We have observed aortic stenosis, dilated cardiomyopathy,
acute myocardial infarction, and extensive coronary artery
disease in El Bagre-EPF patients.
In Chagas disease, similar cardiac dysfunction may occur
as observed in the El Bagre-EPF patients.24 The parasite
T. cruzi is transmitted by insect vectors and is found in rural
areas of Latin and South America. Previous searches for
T. cruzi in the El Bagre area have been positive. We tested
for this parasite in all of the study subjects by blood smear examination, and all tests were negative.
Conclusion
In the current study, we documented for the first time the
expression of MYZAP in cardiac nodes. We documented
that one-half of the El Bagre-EPF patients presented with sinus bradycardia. We described autoantibodies to the cardiac
nodes in patients affected by El Bagre EPF that colocalized
with DPI/II, MYZAP, ARVCF, and p0071 and were located
in the cardiac conduction system. The autoantibodies, complement, fibrinogen, proteases, and their inhibitors seem to
be producing silent clinical abnormalities and ECG alterations, possibly by direct deposition and/or by inducing associated inflammatory molecules. These pathologic changes
directly damage the cardiac nodes and the cardiac conduction
system, and/or they alter cell signaling in the cardiac conduction system. Further study of these autoantibodies presents an
outstanding opportunity to understand cardiac rhythm abnormalities in autoimmune diseases.
Acknowledgment
The authors thank Jonathan Jones, HT (ASCP) at GDA for
excellent technical assistance, and our patients and controls.
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Appendix
Supplementary data
12.
Supplementary data associated with this article can be found
in the online version at https://doi.org/10.1016/j.hrthm.2
017.12.023.
13.
14.
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