Congenital hear

7/27/2019 Congenital hear

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Scuola Superiore di Studi Universitari e Perfezionamento SantAnna

Classe Accademica d i Scienze Sper imenta l i SETTORE DI SCIENZE MEDICHE

CAV3.1T-TYPE CALCIUM CHANNEL AS TARGET

OF CONGENITAL HEART BLOCK-INDUCING AUTOANTIBODIES

STATE OF THE ART AND NEW TRENDS IN CONGENITAL HEART BLOCK ETIOLOGY

ALLIEVO: TUTOR:

FILIPPOQUATTRONE ANTONIOL ABBATE

ANNO ACCADEMICO2010/2011


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Cav3.1 T -type calcium channel as target of CHB-inducing autoantibodies 2

ABSTRACT

Congenital heart block is a disease characterized by impairment of atrioventri-

cular node electric conduction in neonates without cardiac malformations.

The disease is associated with maternal serum autoantibodies against the pro-

tein Ro52. These are thought to cross the placenta barrier and bind to still un-known targets on the cardiomyocyte membrane triggering apoptosis and, in

predisposed fetuses, atrioventricular node fibrosis.

Ro52 has been demonstrated on cardiomyocyte surface only during apoptosis,

and therefore would only be a late target for maternal Ro52 antibodies.

Reduction of L- type calcium currents in cardiomyocytes treated with antibo-

dies from mothers with children affected by congenital heart block suggest vol-tage gated calcium channels as plausible targets in the disease. Particularly the

L- type calcium channels Cav1.2 and Cav1.3 were proposed, even although not

conclusively demonstrated, to be targets.

Now also Cav 3.1, a T type low voltage activated calcium channel, is under in-

vestigation as a possible target for congenital heart block development. Many

clues support this hypothesis such as reduction of T-type calcium currents after

treatments with pathogenic antibodies; the fetal atrioventricular node specifici-

ty of Cav 3.1; the impairment of atrioventricular conduction in its knockout

model, its recognition by sera of mothers of children with heart block.

The presence of multiple targets for Ro52 antibodies in fetal heart could

represent an intriguing hypothesis to explain the complex onset and wide range

of symptoms of congenital heart block.


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CONTENTS

ABSTRACT 2

ABBREVIATIONS 5

INTRODUCTION 6

1 CONGENITAL HEART BLOCK:AN OVERVIEW 7

1.1 The heart block in the newborn and its classification 7

1.2 Isolated congenital heart block as a passively acquired autoimmune disease 9

1.3 Congenital heart block as a multifactorial disease 13

2 A CRITICAL REVIEW OF PROPOSED TARGETS FOR CHB-INDUCING ANTIBODIES14

2.1 The ideal feature of a target for CHB-inducing antibodies 14

2.2 Ro and La antigen surface expression: the apoptosis hypothesis 15

2.3 The serotoninergic hypothesis 17

2.4 The calcium channel hypothesis: the L-type channels 18

3 Cav 3.1AS CHB-ANTIBODY TARGET:CLUES AND PRELIMINARY DATA23

3.1 The Cav 3.1 T-type calcium channel in the heart: structure and functions 23

3.3 Clues in support of a role of Cav 3.1 in CHB 24

3.2 Cav3.1 Hypothesis: preliminary data 26


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CONTENTS

4 AMULTIPLE HIT MODEL FOR CHB:A SPECULATIVE HYPOTHESIS27

CONCLUSIONS 29

REFERENCES 31

INDEX OF FIGURES

Figure 1. ECGs and fetal echocardiographies of different degrees of AV block 8

Figure 2. Current model of CHB and trends of research 13

Figure 3. Cellular topology of Ro and La in cultured human fetal cardiocytes 16

Figure 4. Apoptotic cells are present only in fetal heart conduction system 16

Figure 5. Cross-reactivity of positive IgG with the 1c subunit of Cav1.2 21

Figure 6. Cross-reactivity of positive IgG with the 1d subunit of Cav1.3 22

Figure 7. ELISA results of 161 sera against the E1 loop of the 1D subunit of Cav1.3 22

Figure 8. Effect of CHB inducing IgGs on T-type calcium current 25

Figure 9. ELISA results in support of Cav 3.1 role in CHB 26

Figure 10. A two-stage model of CHB 28


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INTRODUCTION

Congenital heart block appears as an elusive disease: despite that it has beenrecognized as a distinct clinical entity for 100 years very little has been unders-tood about the molecular bases and the development of this condition rare butwith lifelong consequences.

Studying congenital heart block during my two month internship at theHospital for Sick Children of Torontoin Doctor Robert Hamiltons laboratoryunder the supervision of the research associate Linn Strandberg I had the op-portunity not only to practice and watch a wide range of laboratory techniques but also to be involved as an active member in the process of the research. Thishelped me to overcome the initial disorientation I experienced, leading to mydiscovering the satisfactions given by the study of such an unexplored field.

This essay will try to discuss the state of the art and new perspectives in thefield of the individuation of the targets of congenital heart block-inducing antibodies. Especially it will focus on a new candidate target recently proposed byHamiltons laboratory: the T-type calcium channel Cav 3.1. Most part of the ex-periments about this topic, however, is still unpublished and confidential,therefore only some persuasive considerations and preliminary data can be pre-sented in support of the thesis.

The first section of the essay will summarize the main peculiarities of the dis-ease and the different trends of research in this field. The next section will try tocritically analyze the different hypotheses proposed in literature on the natureof the target of congenital heart block-inducing antibody. Afterward attentionwill be conferred to the clues and preliminary data that led to research aboutthe role of Cav 3.1 T- type calcium channel in congenital heart block pathogene-

sis. Eventually a multiple hits model will be proposed as a new perspective toexplain the different symptoms and degrees of the disease.


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1 CONGENITAL HEART BLOCK: A GENERAL OVERVIEW

1.1 THE HEART BLOCK IN THE NEWBORN AND ITS CLASSIFICATION

The congenital heart block (CHB) or congenital atrioventricular block (AVB) is a

pediatric disease, first reported by Morquio in 1901, characterized by various

degrees of impairment of the atrioventricular conduction detectable at birth or

from mid-gestation with the use of diagnostic methods as fetal electrocardio-

graphy (ECG), echocardiography or magnetocardiography (Sonesson 2010).

The AVB is usually classified in three different degrees (see figure 1):

In first-degree AVB, all impulses pass through the atrioventricular (AV)

node, but the AV conduction time is longer than 0,20 s.

Second degree AVB presents a failure to conduct some atrial impulses to the

ventricles. It has three subclassification: the Mobitz type I (Wenckebach) AVB

with a progressive lengthening of the PQ interval and shortening of RR intervaluntil an isolated impulse is blocked; the Mobitz type II AVB characterized by a

sudden block without prior lengthening of the AV conduction time; 2:1 or 3:1

second-degree AVB in which every second or third atrial impulse is conducted

to the ventricles. 3:2, 4:3 or even more complex patterns are also possible.

Third-degree AVB or complete atrioventricular block (CAVB) denotes a situ-

ation where there is no AV conduction at all, and the atria and ventricles beatindependently. (Vijayaraman and Ellenbogen, 2010).

Approximately an half of congenital complete blocks are observed in patients

with complex congenital heart malformations such as abnormalities of the great

arteries, left atria isomerism, atrioventricular septal defects (Machadoet al.,

1988), but approximately one out of every 15 000 to 20 000 births results in a ba-

by with isolated AV block (Buyon, 1998).


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\

Figure 1. ECGs and fetal echocardiographies of different degrees of atrioventricular (AV) block

A) (left)V1 lead ECG of first degree AV block. The PR time is more than 0,20 ms.

(right)Doppler flow velocity recording from the mitral valve (upward) and left ventricular outflow to the aorta (downward)

from a 19 weeks fetus with first-degree AV block. The first vertical line shows the intersection of the mitral early fillingwave (e) and the wave due to atrial contraction (a*); the second line demonstrates the onset of ventricular ejection (v).The AV time interval, in this case more than four z-scores above normal mean, is measured between these two lines.

B) V1 lead ECG of second degree atrioventricular block Mobitz I/Wenckebach. Notice the decrease of RR intervals untilpause.

C) V1 lead ECG of second degree atrioventricular block Mobitz II. Suddenly a P wave is not followed by a QRS complex.

D) (left)V1 lead ECG of 2:1 AV block. Every second P wave is followed by a QRS complex.

(right)A Doppler flow velocity recording from the pulmonary trunk of a fetus with 2:1 AV block. Notice the pattern of thewaves due to atrial (a) and ventricular (v) contractions.

E) (left)V1 lead ECG of a complete, third degree AV block. P waves are independent from QRS complex that follows itsown ventricular pacemaker.

(right) A Doppler flow velocity recording from the pulmonary trunk of a fetus with complete AV block. Indirect markers of atrial (a) and ventricular (v) contraction do not show any constant relationship.

(modified from Sonesson, 2010)

B.

A.

E.

C.

D.

R P RR R R


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1.2 ISOLATED CONGENITAL HEART BLOCKAS A PASSIVELY ACQUIRED AUTOIMMUNE DISEASE

Isolated CHB and maternal autoimmune disease were first associated by Ayl-ward in 1928. Since then CHB has been recognized as a symptom of the neonat-

al lupus erythematosus syndrome (NLE) (Chameideset al. , 1977).

NLE is a passively acquired autoimmune disease that is to say an autoim-

mune disease in which the autoantibodies are not produced by the affected sub-

ject. In NLE maternal antinuclear autoantibodies against the Ro52 and Ro60 an-

tigen (usually defined in clinical as Anti-Ro/SSA) and La (Anti-La/SSB), typicalof Sjgren's syndrome and lupus but also present in asymptomatic patients,

cross the placenta barrier and cause skin rash, hemolytic anemia, leukopenia,

thrombocytopenia, cholestatic liver disease and, the only lifelong symptom,

CHB (Brucatoet al., 2008).

In the last decade also more general effects on the heart were associated with

Anti-Ro antibodies and CHB such as bradycardia, myocardial inflammation,QT interval prolongation, endocardial fibroelastosis and dilated cardiomyopa-

thy, heart failure (Jaeggiet al., 2002). Further support for a spectrum of conduc-

tion abnormalities that extends beyond the AV node comes from autopsies

showing calcification of the sino atrial node (SAN) in CHB human foetal heart

explaining the reported cases of bradycardia (Litseyet al.,1985).

The antibodies against the protein Ro52 are the more related with CHB (Sa-

lomonssonet al., 2002), while anti-La antibodies are higher in cutaneous NLE

rather than CHB (Silvermanet al., 1995).Particularly the antibodies against the

peptide p200 of the Ro52 protein (amino acid 200239) were demonstrated to be

a good biomarker of the disease (Salomonsonet al., 2002; Strandberget al.,

2008).

The binding of the maternal antibodies to cardiomyocytes is thought to cause

first apoptosis then, because of immunocomplexes, the production of profibrot-


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ic cytokines such as TGF-(tumor growth factor) by macrophages. Activation

of TGF- is also mediated by the urokinase plasminogen activator (uPA)/uPA

receptor system over-activated by Ro antibodies. TGF-induces the fibroblastto differentiate in myofibroblasts with a scarring phenotype. Moreover surface

binding by anti-SSA Ro and anti -SSB La antibodies inhibits the uptake of

apoptotic cells by the healthy cardiocytes. This result is accumulation of apop-

totic cells promoting further inflammation and subsequent scarring (Clancy and

Buyon, 2004; Clancyet al. , 2006; Briassouliet al., 2011).

The progression of the disease is actually extremely complex: it is normallydetected between the 18th and 24th week of gestation (Warhen-Herlenius and

Sonesson, 2006), but not all the cases of first degree AV blocks resolve in higher

level block and spontaneous reversibility of first degree block is quite common

(Jaeggiet al., 2011). On the contrary, a first degree CHB was reported to

progress to a CAVB even after the postnatal clearance of maternal antibodies

(Askanaseet al., 2002).Transplacental treatment with fluorinated steroids inhibit and sometimes re-

verse the progression of the disease, but has reported side effects (Cavalieriet

al. , 2006). A CAVB once occurred is a permanent situation treatable only with

long life heart pacing. (Boutjdir, 2000).


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1.3 CONGENITAL HEART BLOCK AS A MULTIFACTORIAL DISEASE

The presence of anti-Ro/SSA or La/SSB in the maternal sera does not automati-

cally lead to NLE or CHB and actually only 2% of mothers with the anti-Ro 52antibodies will result in affected pregnancies (Brucatoet al., 2001; Cimazet al.,

2003).

The reason of these low rate of affected pregnancies in high risk subjects is

due to the still not understood etiopathogenesis of the disease. A complex pat-

tern of genetical, epigenetical, and environmental factors are thought to take

part in the development of CHB. This view is also supported by the low rate ofrecurrence (15%) of CHB in subsequent pregnancies (Buyonet al., 1998) and the

report of discordance for CHB in monozygotic twin pairs (Buyonet al.,1998).

Due to the likely role of an inflammatory process in the establishment of the

atrioventricular node block and fibrosis efforts were made to discover an asso-

ciation between CHB and HLA (human leukocyte antigen) genes in the mother

or children. Studies with Ro52 immunized murine models demonstrated that

generation of pathogenic Ro52 antibodies is restricted by maternal MHC (major

histocompatibility complex), whereas the fetal MHC locus regulates susceptibil-

ity and determines the fetal disease outcome (Strandberget al., 2010).

A parental effect was demonstrated on the susceptibility to heart block since

maternal origin of certain MHC alleles correspond to significantly longer PR in-

tervals than paternal inheritance of the same alleles (Strandberg, 2007). No con-

clusive evidence of a HLA and CHB association were found yet in humans even

if there were different proposals. (Sirenet al., 1999).

Non-HLA genes have also been suggested to play a role in CHB susceptibili-

ty. It was proposed an association of CHB with TGF-and tumor necrosis factor

polymorphisms as there was an increased frequency of cytokine polymor-


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phisms in 40 children with CHB compared to a large number of healthy con-

trols (Clancyet al., 2003).

Another phenomenon studied to explain the complex inheritance of CHBand particularly the discordance between twins was maternal or sibling micro-

chimerism, i.e. the presence in an host organism of a small number of cells that

originated from another individual. Maternal myocardial cells have been found

in the hearts of patients with CHB (Stevenset al., 2003), suggesting that mater-

nal microchimerism may play a role in CHB development but the correlation

between detection of maternal and/or sibling microchimerism and CHB couldnot be universally established (Stevenset al., 2005).

Not only the presence, but also the levels of maternal anti-Ro52 antibodies

can play a role in the fetal outcome, as suggested by the recent findings that

CHB is associated only with high or moderate levels of Ro antibodies (Jaeggiet

al., 2010).

In these years researchers are trying to organize in a coherent model the dif-ferent results obtained in CHB studies (see figure 2). In this effort the characte-

rization of the fetal heart CHB autoantibodies target is considered a major step

forward (Warhen-Herlenius and Sonesson, 2006). This discovery, actually, not

only will provide us with a further comprehension of the disease but will also

have important clinical consequences as the development of ELISA (enzyme

linked immunosorbent assay) tests for high risk pregnancies and of therapeuticdecoy peptides that, given during pregnancy, could bind to the pathogenic ma-

ternal antibodies (Carnabiet al., 2010).


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Mother with CHB-inducingautoantibodies

CHB-inducing antibodiesenter fetal circulation

CHB-inducing antibodiescross-react with a target specific

to the fetal AV node

The binding with one or more surfacetargets induces cell death and a

inflammatory response that leads to

CONGENITAL HEART BLOCK

Characterization of CHB inducingmaternal antibody

Study of the genetic, epigenitic,environmental risk factors

TRENDS OF RESEARCH

CHARACTERIZATION OF THE TARGETOF THE CHB-INDUCING ANTIBODIES

Study of the pathogenesis of thedisease

Early detection of the AV blockand treatment with anti

inflammatory drugs

A.

B.

C.

D.

Figure 2. The current model of congenital heart block (CHB) and the research trends in this field

A)

CHB is associated with presence of autoantibodies in maternal sera. Great efforts were made to iden-tify the antibodies causing the disease to develop tests to identify high risk pregnancies. Another trendof research in the field of the prevention of the disease is the improvement of AV block prenatal detec-tion and the study of the effectiveness of the anti inflammatory drugs against the progression of theblock

B) The maternal autoantibodies have to cross the placenta barrier and enter fetal circulation to trigger thedisease. Genetic, epigenetic, and environmental factors promoting the onset of the disease are stilldebated and object of study.

C) CHB inducing antibodies target a surface epitope on cardiomyocytes surface. The individuation of thistarget, that should be specific to the AV, will be a fundamental step in our comprehension of the dis-ease and could open the opportunity to develop peptides to bind maternal antibody before they cross

the placenta.D) The culprit antibodies bind to a extracellular domain of one or more protein triggering cell death. The

individuation of the target (or targets) of pathogenic antibodies should be the first step to deeper un-derstand the etiopathogenesis of the disease clarifying the reason of cell death and the path that leadsto the fibrosis of the atrioventricular node.

(Image of the author)


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2 A CRITICAL REVIEW OF PROPOSED TARGETS FORCHB-INDUCING ANTIBODIES

2.1 THE IDEAL FEATURE OF A TARGET FOR CHB-INDUCING ANTIBODIES

Before analyzing the current candidate target for CHB-inducing antibodies some

characteristics they should satisfy will be listed.

First of all the disease is characterized by block only at the level of the AV node,

therefore the antibody target should be only or mostly expressed in the AV node.

On the other hand the candidate should also explain the other symptoms recentlyrelated to the AVB especially the bradycardia often present in children exposed to

maternal autoantibodies (Brucatoet al., 2000).

Also, the identified target should clarify why mothers with affected children do

not show AV block. This could be due to a fetal unique isoform or a post-

translationally modified form (e.g. a fetal specific pattern of glycosilation)of the

protein targeted by maternal antibodies. Recently it was shown a prolongation of

corrected QT interval also in adults with anti Ro52 antibodies (Lazzeriniet al. , 2010,

Lazzeriniet al., 2011b). This data could account for a resistance of adult heart

against the antibodies-mediated damage rather than a fetal specificity of the target

(Lazzeriniet al. , 2011a).

Moreover the target should be proven with different laboratory techniques such

as indirect ELISA and immunoprecipitation to prove maternal sera reactivity with

the target. Ideally, one should also be able to prove an alignment of a segment ofthe target sequence with the p200 peptide of Ro52 however the p200 epitope has

been demonstrated to be three dimensional (Ottossonet al. , 2005), then sequence

alignments may not be possible. Eventually the immunological insult mediated by

the antibodies against the target should be proven as the start point of the cascade

that leads to the fibrosis. This could be achieved within vitro methods studying

cultured cardiomyocytes treated with antibodies against the target, as well as within vivo immunization animal models.


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2.2 THE APOPTOSIS HYPOTHESIS

Despite the well established association of anti-Ro and anti-La antibodies with NLE

and CHB, it is debated if their antigens play a role in the onset of the diseases.

The problem is that Ro52, Ro60 and La are intracellular proteins and therefore

not accessible to circulating maternal antibodies. The apoptosis hypothesis fore-

sees that anti-Ro and anti- La antibodies are translocated to the cell surface during

apoptosis, where they are bound by maternal antibodies. This is supposed to in-

duce engulfment of the apoptotic bodies by macrophages through opsonization

with production of pro-inflammatory and pro-fibrotic cytokines, recruitment ofleukocytes and complement components leading to an inflammatory reaction that

damages the AV node (Miranda-Caruset al., 2000; Clancyet al., 2004).

The first evidence of exposure of Ro and La in apoptotic bodies was found in

coltured keratinocytes (Casciola-Rosenet al.,1994). This finding was then confirmed

in human and mouse heart fetal cardiomyocytes with immunofluorence and elec-

tromicroscopy, but not yetin vivo (Miranda-Carset al., 1998; Miranda-Carset al.,2000, Tranet al. , 2002 ) (see figure 3).

The apoptosis hypothesis does not however explain the specificity of the reac-

tion in targeting the heart and in particular the AV node. Moreover it does not ac-

count for the direct electrophysiological effects of maternal anti-Ro/La autoantibo-

dies on cardiomiocytes in patch clamp studies. Furthermore as previously said

transient first-degree AVB is often detected in fetuses exposed to Ro antibodies

(Sonessonet al., 2004), indicating a direct effect of maternal antibodies without in-

duction of self-amplifying cardiac inflammation as implied in the theory.

The apoptosis hypothesis can give, instead, explanation to the age-dependence

of CHB because no apoptosis was detected in the healthy adult fetal heart conduc-

tion system and working myocardium despite its presence in fetal heart (Tranet al. ,

2002; see figure 4).


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E

Figure 4 Apoptotic cells are present only in fetal heart conduction system

A) Low-power (scales 100 m) confocal images of afetal heart at 15 days gestation dual-labeled for apoptosis by TUNEL (green) and DNA(PI, red), showing TUNEL-stained apoptotic cells within and around the atrioventricular (AV) node.

B) H&E staining of the same section shown in A, indicating the location of the AV node (AVN); interatrial septum (IAS); interventricular septu

(IVS); superior vena cava (SVC); left atrium (LA); right atrium (RA); root of the tricuspid valve (TVR). Inset, the characteristic condensnucleus of the apoptotic cell (arrowhead), corresponding to TUNEL labeling in A.

C) No TUNEL-positive cells are seen in the low-power (scales 100m) confocal image of the adult mouse heart.

D) H&E staining of the same section shown in C, indicating the location of AV node and the other structure indicated in B.

(modified from Miranda-Cars et al., 1998 and Miranda-Cars et al., 2000)

C D

Figure 3. Cellular topology of Ro and La in cultured human fetal cardiocytes

A-D Immunofluorescent staining of cultured fetal cardiocytes. Surface stain was made with Anti- Ro or Anti La sera (green), and nuclear stainingwith propidium iodide (PI, red). The images of apoptotic cell were taken 6 h after induction of apoptosis with 0.5 mM staurosporine. InA nonapoptotic fetal heart cell stained with Anti-La antibodies. La is found principally in nuclei (the combination of green and red stain gives a yel-low signal). In B apoptotic fetal heart cell stained with Anti-La antibodies.. La translocted from nucleus and concentrates in apoptotic blebs. In C non apoptotic fetal heart cell stained with Anti-Ro antibodies. Ro antigen is more concentrated in nuclei. InD Ro is seen in the nucleusand cytoplasm of non apoptotic cells. There is translocation of Ro to the cell periphery and staining of blebs.

E-G Scanning electron micrographs of human fetal cardiocytes labeled with immunogold attached to a serum containing Anti-Ro and Anti-La antibodies. In E non apoptotic cardiomyocyte (notice the smooth surface) does not present attached gold particles, therefore no Ro or La anti-

gens are normally exposed on their surface; InF early apoptotic cardiomyocyte (rough surface) presents antibodies attached to their surface(white dots); InG also late apoptotic cardiomyocyte (notice the emerging blebs) presents antibodies attached to their surface (white dots).

(modified from Miranda-Cars et al., 1998 and Miranda-Cars et al., 2000)

A B C D

FE G


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2.3 THE SEROTONINERGIC HYPOTHESIS

Another school of thought in CHB etiology believes that Anti-Ro does not bind

their cognate target in fetal heart but they cross-react with a surface target, in-ducing cell death and impairment of atrioventricular conduction.

One target proposed was the serotoninergic 5-HT4 receptor(Eftekhariet al.

2000), the only metabotropic receptor bound by serotonin.

The original study showed that antibodies to the Ro52 peptide 365-382 rec-

ognized residues 165-185 of the cardiac 5-HT4 receptor corresponding to the

second extracellular loop, the immunodominant region of the protein, and that

affinity-purified anti-5-HT4 antibodies could antagonize the serotonin-induced

calcium channel activation in atrial cells (Eftekhariet al., 2000). However mouse

pups born to females immunized with Ro52 peptides that had been selected on

the basis of recognition by anti-5-HT4 antibodies did not develop any sign of

AV block (Eftekhariet al., 2001). Moreover the Ro52 peptide 365-382 has not

been demonstrated to be associated with CHB (Salomonssonet al., 2002).

In a later study by the same authors, only 16% of the sera from mothers of

children with CHB were shown to be positive for anti-5-HT4 antibodies, indi-

cating that cross-reactivity to the serotoninergic 5-HT4 receptor, if indeed in-

volved in the development of CHB, may only represent a small subset of cases

(Kamelet al., 2005).

Moreover the serotoninergic hypothesis does not account for the specificity

in AV node, but it could account for age-dependence since it was reported that

in adult mouse heart, the 5-HT4 receptor, although expressed, is not functional-

ly coupled to the transduction pathway (Ouadidet al., 1992).


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2.4 THE CALCIUM BLOCKADE HYPOTHESIS: L-TYPE CHANNELS AS TARGET OF CHB-INDUCING ANTIBODY

The first evidence ofcalcium homeostasiss involvement in CHB etiology camefrom studies about the arrhythmogenicity of antibodies from sera of mothers of

children with CHB. The experiments, indeed, showed that IgG-enriched frac-

tions and anti-52-kD SSA/Ro affinity-purified antibodies from maternal sera in-

duced CAVB in rat and human fetal heart perfused by the Langendorff tech-

nique and inhibited L-type calcium currents (ICa-L) at the whole-cell and single

channel level (Boutjdiret al. , 1997;Boutjdiret al.1998).To study this phenomenon researchers tried to identify which L type channel

was affected by antibody exposure. Two L-type channels are principally ex-

pressed in the heart: Cav1.2 (called also1C from the name of its ion conducting

pore subunits) and Cav1.3 (called also1D). Cav1.2 is ubiquitously expressed in

the heart and mediates excitationcontraction coupling. Cav1.3 is highly ex-

pressed in the SAN and AV node (Mangoniet al., 2003; Zhanget al. 2002).Cav1.2 Ca current activates at 40 to 30 mV and accounts for the electrogenesis

of the action potential at the AV node, whereas Cav1.3 Ca current activates be-

tween 60 and 40 mV, at a range corresponding to the diastolic depolarization

in the SAN(Irisawaet al., 1993).

Understand which channel is bound by maternal antibody is very challeng-

ing because both Cav1.2 and Cav1.3 Ca channels contribute to the total ICa-Land both are sensitive to the Ca channel blockers. Moreover the embryonic lethality

of the Cav1.2 knockout mouse model limited the use of transgenic mouse mod-

els (Seisenbergeret al., 2000) to study isolated Cav1.3 ICa-L. Therefore the study of

Cav1.3 ICa-Lhas been limited to expression systems such as tsA201 cells (a sub-

clone of the human embryonic kidney cell line 293 that expresses simian virus

40 T antigen) that do not express endogenous Ca channels. Only recently a nov-el model of effective lentiviral silencing of Cav1.2 allowed for the investigation


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and characterization of the Cav1.3 Ca channels in native cardiomyocytes (Kar-

nabiet al., 2009).

The first studies concentrated to Cav1.2. The first direct evidence of reaction between the channel and the maternal antibodies was obtained inXenopus oo-

cytes treated with immunoglobulins G (IGGs) from mother with CHB affected

children after transfection with the channel cRNA. The study showed signifi-

cant reduction of L-type current in oocytes expressing the1c pore forming

subunity proving a direct binding with the principal subunity of the channel.

(Xiaoet al., 2001) (See figure 5A). A direct proof of the binding was given byimmunoprecipitation with anti-1C antibody: the protein obtained were sepa-

rated by electrophoresis, transferred to membrane and then probed with anti-

1C antibody, IgG from mother with children with CHB and IgG from a

healthy subject. The products of immunoprecipitation treated with anti-1C an-

tibody and maternal IgG gave two bands at the same level while the healthy

control did not present any band. (See figure 5B)Cav1.3 emerged as a candidate target for maternal CHB-inducing antibody.

studying the bradycardia induced by maternal IgGs. First of all isolated SAN

cells were treated with maternal antibodies inducing, as expected, reduction of

L type (but also T-type) calcium currents not affecting the inward rectifier or the

funny current (Huet al., 2004). Then researchers expressed1D (the pore form-

ing subunit of Cav1.3) in heterologous expression systems (tsA201 cells and Xe-nopus oocytes) and showed inhibition of1D ICa-Lin both expression systems

after treatment with maternal CHB IgGs (Quet al., 2005). (See figure 6A)

Also western blot experiments were performed on Cav1.3 extracted from

transfected tsA201 cells. Anti-1D antibody recognized the 190-kDa band cor-

responding to the channel protein. Then the membrane blot was stripped and

reprobed with positive and negative IgGs, respectively. Positive but not nega-tive IgG recognized the same 190-kDa band (Quet al., 2005). (See figure 6B)


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Eventually a possible binding sites on1D protein for autoantibodies from

sera of mothers with CHB children was proposed (Karnabiet al., 2010). For this

aim proteins of the extracellular regions between the transmembrane segments(S5S6) of each of the four1D Ca channel protein domains were prepared and

tested for reactivity with sera from mothers with CHB children and controls us-

ing ELISA. These protein segments, also called E loop, were chosen because

they form the ion conductance pore and selectivity filter of the channel. Only

the E loop of the first domain of the protein was recognized by maternal sera.

The evidence obtained is, however, very weak and need further confirmations

(see figure 7). Some recently disclosed preliminary data provide the first evi-

dence that the Cav1.3 ICa-Lis inhibited in native rat cardiomyocytes. In this expe-

riment maternal CHB autoantibodies induced inhibition of the Cav1.3 ICa-L by

35% after lentiviral silencing of Cav1.2 (Karnabi and Boutjdir, 2010).

Both acute and chronic consequences of Ca channel blockade by maternal an-

tibodies were proposed. Acute events are initiated by binding of the circulatingmaternal antibodies to L-type Ca channels. This binding account for the elec-

trophysiological alterations seen in the experiments. The chronic presence of an-

tibodyantigen complexes is thought to result in internalization and degrada-

tion by lysozymes of the calcium channels. Cells with enough remaining Ca

channels will survive and secure atrioventricular conduction; those with severe

depletion of Ca channels will result in Ca dysregulation and apoptosis leading

to impairment of heart electric conduction (Karnabi and Boutjdir, 2010).

This view is supported by an experiment made on 2 mouse models: one

transgenic mouse with overexpression of Cav1.2 1C subunit and a mouse

knockout for Cav1.3. Immunization with SSA/Ro and SSB/La antigens of mice

over-expressing Cav1.2 give birth to pups with no or fewer electrocardiographic

abnormalities than wild type ones. On the contrary immunization of Cav

1.3


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knockout mice give birth to pups with higher level of AV block than non im-

munized Cav1.3 knockout mice.

The L-type Calcium channel hypothesis is dominant in the field of congenitalheart block etiology. It has, however, different point of weakness. There are no

studies trying to investigate the specificity of the antibodies binding to the

Cav1.2 or Cav1.3. Moreover no binding site was identified for Cav1.2 and the site

identified for Cav1.3 is not convincing.The difficulties to find the channels ep i-

tope could be due to the channels used in the experiments. Indeed they could

be mysfolded and leaking post-tradutional modifications coming from expres-sion systems that did not guarantee for the of these complex proteins.

Eventually the calcium hypothesis cannot account for the low incidence of

CHB in infants from mothers with Ro antibodies but could explain the invulne-

rability of the adult conduction system cells to anti-Ro/SSA antibodies. Adult

cells, indeed, have a higher calcium channel reserve that could preserve them

from calcium homeostasis dysregulation induced by antibody-mediated inter-nalization of calcium channels (Lazzerini, 2011).

A B

Figure 5. Cross-reactivity of positive IgG with L-type Ca channel with the 1c subunit of Ca v1.2

A) Steady-state effect of positive IgG (350 g/mL) on I-V relations of IBa- 1C in Xenopus oocytes transfected with

1c. Notice the inhibition of the current after the treatment. In this electrophysiological experiment barium is

used as charge carrier instead of calcium.

B) SDS-PAGE analysis of 1C protein immunoprecipitated with a anti-1C antibody (Card I) from transfected oo-cytes. Lane 1 shows reactivity to Card I, lane 2 to positive IgG, and lane 3 to negative IgG. Lane 4 shows reac-

tivity to Card I after blot of lane 3 was stripped (to demonstrate that1C protein was present when subjected to

negative IgG).


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Figure 6. Cross-reactivity of positive IgG with L-type Ca channel with the 1d subunit of Ca v1.3

A) ICa- 1D Lwas inhibited by positive IgG in transfected tsA201 cells. ICa- 1D Lwas recorded with whole-cell mode

of patch-clamp technique with2 mmol/L Ca as charge carrier. A, Dose-response curve of positive. Averaged

I-V curves obtained by depolarizing pulses between 80 and 50 mV from holding potential of 100 mV before

and after application of 100 g/mL positive IgG.

B) (Above)Western blot from 1D protein transfected tsA201 cells was assessed. 1D protein was detected in

all lanes with anti- 1D antibody.(Middle)Anti- 1D antibody was stripped from membrane.(Bottom)half of

the membrane was reprobed with positive IgGs that recognized the same band of 1D antibody. Remaining

half of stripped membrane was reprobed with negative IgG that did not cross-react with1D protein.

(from Qu et al. , 2005)

A B

Figure 7. ELISA results of 161 serum samples against the E1 loop of the 1D L-type Ca channel.

Only seventeen of 118 (14.4%) samples from the CHB group were ELISA positive and above the O.D. cutoff point of 0.1

which represents the average for the healthy sera plus 2 standard deviation. In contrast, sera (with anti-SSA/Ro and

SSB/La antibodies) from mothers with healthy children did not react (0/15) with the E1 loop, while in the sera (without

anti-SSA/Ro and SSB/La antibodies) from mothers with healthy children, only 2 of 18 (7%) were ELISA positive.

(from Karnabi et al. , 2010)


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3 CAV 3.1AS CHB-INDUCING ANTIBODY TARGET: CLUES AND PRELIMINARY DATA

3.1 CAV 3.1 IN THE HEART: STRUCTURE AND FUNCTIONS

Cav3.1 is a voltage-dependent calcium channel belonging to the electrophysio-

logical class of low voltage activated calcium channel and to the pharmacologi-

cal family of T-type calcium channel. Indeed voltage-dependent Ca2+ channels

are electrophysiologically divided into two major classes: low voltage-activated

and high voltage activated Ca channels. Instead according to the pharmacologi-cal and biophysical properties they are subdivided into five groups (P/Q, N, L,

R, and T). Low voltage activated channel class consists of only T-type Ca2+

channels which open at low membrane potentials(30 mV) and inactivate very

rapidly (T stay for tiny or transient current) in contrast to the high voltage acti-

vated L (long-lasting) -type Ca channels discussed above (Vassortet al., 2006;

Ono and Iijima, 2009). Whereas L-type Ca2+ channels are composed of foursubunits 1, 2 , , , only subunit 1 is confirmed for T-type channels even if

many clues suggest coexpression with2 (Walshet al. , 2009; Dubelet al., 2004).

Three different T-type calcium channel were identified: Cav3.1, Cav3.2, Cav3.3.

They have different isoforms of1subunit, respectively1G, 1H, 1I.

In the heart L-type Ca channels are responsible for Ca entry into the cell,

which triggers contraction, while T-type Ca2+ channels contribute significantlyto cardiac automaticity, development and excitationcontraction coupling. Fur-

thermore, they have a role in pathological process such as cardiac hypertrophy

and heart failure (Ono and Iijima, 2009). Cav3.1 and Cav3.2 are the principal

cardiac isoforms although also CaV3.3 mRNA was reported in Purkinje fibers

(Han et al., 2002). Cav3.1 is abundant in fetal and adult heart conduction sys-

tem, whereas Cav3.2 decreases after fetal life (Chandleret al., 2009; Greeneretal. , 2010; Qu and Boutjdir, 2001).


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3.2 CLUES IN SUPPORT OF A ROLE OFCAV 3.1 IN CHB

Cav3.1 is now under research as a possible target for CHB-inducing antibo-

dies. Many elements present in literature would support this hypothesis.

The first clues suggesting involvement of T-type channel in the disease came

from electrophysiological studies. These showed reduction of T-type calcium

currents in SAN cells after treatment with sera of mothers with affected child-

ren (Huet al. 2004) (see figure 8). This induces to think that also T-type calcium

channels are bound by CHB-inducing maternal antibodies.

Cav3.1, moreover, is highly specific to the compact AV node as showed by

quantitative PCR data (Greeneret al., 2010) and it is upregulated during fetal

life (Cuiet al., 2010). Another interesting aspect of this protein is its high num-

ber of splicing isoforms. Studies on this channel from 1,580 fetal and adult hu-

man brain reported about 30 different isoforms with a developmentally regu-

lated splice variant expression (Emericket al.,2006). Besides analysis of pre-

dicted 1G/CaV3.1 amino acid sequences suggests at least eight phosphoryla-

tion sites (Klugbaueret al., 1999) and eight glycosylation sites (Mittmanet

al.,1999) that could be developmentally regulated. The discovery of a splicing

and glycosylation variants specific to the fetal AV node could explain why the

disease is typical of this tissue and the invulnerability of adult heart. Also the

Cav3.1 knockout mouse presents promising feature: it shows bradycardia and

slowing of the AV conduction similar to the observed lengthening of PR inter-val in fetuses before the block have developed.

Only further experiments will directly prove whether the Cav3.1 channel

plays a role in CHB etiopathogenesis. The next section will present some pre-

liminary data from ongoing experiments disclosed in form of abstract in occa-

sion of the "International workshop on clinical and molecular aspects of CHB"

held in 2010 in Stockholm.


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Figure 8. Effect of CHB inducing IgGs on T-type calcium current

A) Dose-response relation for the effect of CHB-inducing IgGs on ICa-T (T-type calcium current ) at 40 mV

from a holding potential of 80 mV.

B) Time course of peak ICa-T at 40 mV from a holding potential of 80 mV in single SA node cells before

and during application of CHB-inducing IgGs (100 g/mL). Original traces during control and positive

IgG are shown in the inset.

C) Averaged current density-voltage relations of peak ICa-T in response to various depolarizing pulsesfrom a holding potential of 80 mV before (control, n 5) and during application of CHB-inducing IgGs

(100 g/mL, n 5). *P=0.05; **P=0.01.

(from Qu et al. , 2004)


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3.3 CAV3.1HYPOTHESIS: PRELIMINARY DATA

To prove involvement of Cav3.1 in CHB a rabbit Langendorff model and an

ELISA test were assessed (Cuiet al., 2010).

II degree AV block was demonstrated in isolated Langendorff rabbit hearts

perfused with an antibody raised by recombinant peptide amino acid (a.a.) 130

385 of 1G. Moreover treatment with Mibefradil, a T-type calcium channel

blocker (against both Cav3.1 or Cav3.2 subunits) demonstrates markedly en-

hanced dose effect of AV block in immature Langendorff rabbit hearts than ma-

ture hearts (whereas a specific blocker for1H has no enhanced effect).

A PEPScreen library (15 a.a. peptides with overlaps of 10 a.a., corresponding

to the a.a. 130385) for 1G was tested for reactivity with sera from mothers

with CHB children and controls using ELISA. Of 130 peptides, peptides 36 and

37 (a.a. 305320 and 310325) demonstrated the highest reactivity with two sera

from mothers with CHB children (figure 9A). Five of 23 (22%) CHB maternal

sera reacted with 1G peptide 36 (a.a. 310325). In contrast, none of 14 (0%) sera

from healthy mothers reacted with this peptide (P < 0.05) (Figure 9B).

Number of peptides (1-50)

Absor bance

Absor

bance

CHB Healthy

Figure 9. ELISA results in support of Cav 3.1 role in CHB

A) Only peptides 36-37 (a.a. 305 320 and 310 325) from a PEPscreen library for 1G reacted with sera from mother

with CHB children.

B) Peptide 36 was recognized by 5 of 23 sera from mother with CHB children while no sera from mothers with healthy

children reacted with this peptide.

(from Chu et al. , 2010)

B A


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4 A MULTIPLE HIT MODEL FOR CHB: A SPECULATIVE HYPOTHESIS

The experiments presented in the previous sections showed how sera frommother with CHB children are likely to react with multiple targets. The re-

searchers are trying to characterize the role of the different targets in CHB eti-

opathogenesis.

In the last years large consensus (Wahren-Herlenius and Sonesson, 2006;

Karnabi and Boutjdir, 2010) have received a two-stage model of the disease: the

crossreacting target (calcium channels and serotoninergic receptor) wouldrepresent the first step of the disease triggeringthe cardiomyocytes apo ptosis

due to alteration of calcium homeostasis mediated by internalization and diges-

tion of calcium channels. During apoptosis Ro and La antigens are exposed on

apoptotic blebs surface where they are bound by maternal antibodies (second

stage). The inflammation due to the immunocomplexes leads eventually to AV

node fibrosis and complete conduction block (See figure 10). This model is,however, far from being provenin vivo.

Beyond this two-stage model is now emerging a multiple-hit model to cha-

racterize at molecular level the different symptoms of the disease. For example

Cav1.2 because of its presence in the ventricular muscle and its role in electro-

mechanical coupling was proposed to be responsible of the heart failure present

in about 10% of children with CHB (Karnabi and Boutjdir, 2010). Cav1.3 wasprincipally studied in SAN and it was correlated with the bradycardia present

in a large amount of patients (Quet al , 2004). The Cav3.1 channels, eventually,

could be the principle responsible of pathology in AV node and real trigger of

AV block.


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Figure 10 A two-stage model of CHB.

The first stage is initiated by binding of the circulating maternal antibodies to surface L-type Ca channels and 5-HT4

receptor (step 1). As a result of the interaction, the surface complex is internalized and degraded by lysozymes (step

2). In step 3 and 3a, two scenarios are proposed: fotal cells with enough remaining Ca channels will survive and se-

cure atrioventricular conduction; those with severe depletion of Ca channels (step 4) will result in Ca dysregulation

and apoptosis (step 5). This may lead to the translocation of the intracellular SSA Ro and SSB La to the cellsurface

to bind to their cognate antibodies (step 5) increasing the number of surface immunocomplexes. Altogether, this

process will eventually trigger inflammation (step 6), cell death, fibrosis and calcification.

From Karnabi and Boutjdir, 2010


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CONCLUSIONS

The characterization of the targets of CHB-inducing antibodies in fetal heart has

always been considered an issue of first importance towards a further compre-

hension of the disease. However few laboratories have explored this field and

often were not able to prove their theses. This could be ascribed to different

mistakes in the assessment of their experiments. For example, the use of expres-

sion system (oocytes, tsA2019 cells) instead of fetal heart samples is not reliable

because such cells cannot guarantee correct post-tradutional maturation of

channels. A correct folding and glycosylation, however, are essential to analyze

the conformational epitopes probably recognized by Ro52 antibodies. Not only

the expression systems can give bad results but also the use of animal samples

because there are no natural models of the disease and also experimental ani-

mal models are not satisfactory. Moreover many studies proposed targets with-

out characterizing in maternal sera the pathogenic antibody specificities or the

site on the protein recognized by these antibodies.

Progresses in this field would be obtained by merging the results obtained by

different trends of research on CHB. One of the most promising achievement of

the last years was the correlation of the antibodies against the p200 peptide of

Ro52 and the disease. Of great interest would be to investigate if Ro52 p200 an-

tibodies bind to Cav3.1 or another target in fetal heart. This could be obtained

immunoprecipitating Cav3.1 or another target in fetal heart with a specific

commercial antibody and then proving if the product is recognized by maternal

sera and Ro52 p200 antibodies. Another interesting experiment to confirm a pa-

thogenic role for p200 antibodies could be the use of the confocal microscopy to


30/35


confirm if Ro52 p200 antibodies bind to heart cell surface. Also patch clamp and

Langendorff experiments could be assessed to study alteration of calcium cur-

rents and heart conduction after treatment with Ro52 p200 antibodies.Then it should be studied why fetal AV node is the most sensible part of the

heart. This could be achieved studying the transcript variants of Cav3.1 and of

the other targets in fetal AV node regions using trascriptome analysis and real

time PCR. Also glycosylation pattern should be considered in the analysis of

fetal AV peculiarities.

Another important goal is to develop diagnostic and prognostic tool to detecthigh risk pregnancies. Cav3.1 calcium channel peptide ELISA assay could

represent such clinical tool. Also protoarray technology could be used to identi-

fy further epitopes or mimetopes (a peptide which mimics the structure of an

epitope) of antibody reactivity which can be used to develop clinically useful

biomarker assay. The use of protoarray could also very useful to prove the

presence of multiple targets.The development of prognostic tools should have the aim to prevent the on-

set of the block. After the characterization of the target different strategies could

be used to avoid AV node damage such as the use of decoy peptides against

culprit antibodies or plasmapheresis.

Knowledge of the fetal heart target and pathogenic antibody specificity could

also help to make the current anti inflammatory therapies less dangerous, re-

stricting their use only in really at risk pregnancies and only for the period of

expression of the target. Moreover the theory of calcium channels internaliza-

tion, if proven, would suggest Ca channel agonists as a potential prognostic

tools to prevent the calcium dysregulation and subsequent cardiomiocytes

apoptosis.


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