Evidence of anti-cardiac antibody deposition in end-stage failing myocardium: A

potential contributor to disease progression

Christian Assad-Kottner, MD1, 2; Andrea M Cordero-Reyes, MD2; Alejandro R Trevino, MD2; Jose H Flores-Arredondo, MD2; Evaristo Fernandez2, MD; Roberto Barrios, MD2; Keith A Youker, PhD2; Guillermo Torre-Amione, MD, PhD2

The University of Texas Medical Branch at Galveston Texas (UTMB)The Methodist DeBakey Heart Center

Herskowitz, A. et al. (1993) Concepts of autoimmunity applied to idiopathic dilated cardiomyopathy. J. Am. Coll. Cardiol. 22, 1385–1388

IntroductionSeveral studies have suggested the possibility that the humoral immune response may play an important roll in the development and progression of heart failure.

Previous studies have focused on demonstrating the presence of antibodies against the myocardium in the serum of patients with cardiomyopathy.

To date, no study has shown the deposition of native anti cardiac antibodies deposited in the patient's failing myocardium

Antibodies

Mitochondria

M7ANT

BCKD-E2

Sarcolemma / Contractile

Proteins

TroponinActin

Myosin

MuscarinicReceptors

B1-AdrenérgicReceptors

Caforio AL, Mahon NJ, Tona F, McKenna WJ. Circulating cardiac autoantibodies in dilated cardiomyopathy and myocarditis: pathogenetic and clinical significance. Eur J Heart Fail. 2002 Aug;4(4):411-7.

Introduction

Introduction

The presence of anticardiac autoantibodies have been studied in the serum of patients with heart failure since 1986.

One of the biggest challenges up to date regarding the presence of anticardiac antibodies has been verifying their specificity since a great number have been shown to be present in similar numbers in healthy individuals.

Schimke I, Muller J, Priem F, et al. Decreased oxidative stress in patients with idiopathic dilated cardiomyopathy one year after immunoglobulin adsorption. J Am Coll Cardiol. 2001;38:178–183.

Several groups have shown that removing anticardiac antibodies in patients with heart failure improve systolic function, diastolic function and NYHA class.

When removing these antibodies particular importance has been placed in the IgG3 subclass.

Introduction

IntroductionHow where the Anticardiac Antibodies removed?

Plasmapheresis

Inmunoadsorption

Felix SB, Staudt A. Immunoadsorption as treatment option in dilated cardiomyopathy. Autoimmunity. 2008 Sep;41(6):484-9.

So… What is so special of IgG3Activates the complemente in the most efficient way

From all the subclasses it is the one that has the most notable cytotoxic activity

Several autoimmune disease have shown to have IgG3 as a predmominant subtype (Rheumatoid Arthritis, Connective Tissue Diseases, Psoriasis)

Introduction

Staudt A, Böhm M,Felix SB. Potential role of autoantibodies belonging to the immunoglobulin G-3 subclass in cardiac dysfunction among patients with dilated cardiomyopathy. Staudt A, Böhm M,Felix SB. Circulation. 2002 Nov 5;106(19):2448-53.

Jahns R, Boivin V, Siegmund C, et al. Autoantibodies activating human B-1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure. Circulation. 1999;99:649–654

Antibodies against the B-1 Adrenoreceptor

60% of patients with idiopathic cardiomyopathy have it<1% of healthy individuals10-13% of patients with ischemic cardiomyopathy

Introduction

MethodsMyocardial samples were obtained from 100 patients with heart failure at the time of transplantation or implantation of a left ventricular assist device.

40 samples from normal hearts were used as control

Samples were then fixed in paraffin following standardized protocols

MethodsThe samples were subsequently analyzed by immunofluorescence techniques in order to identify; 1) Anticardiac antibodies 2) The IgG3 subclass 3) Activated Complement

Antihuman antibodies conjugated with FITC (fluorescein isothiocyanate) or Cy3 (carbocyanine 3) were used to identify native anticardiac antibodies and activated complement.

Samples were then analyzed by a board certified pathologist

Antibodies 1

01

IgA IgD IgE IgG IgM

IgG1 IgG2 IgG4IgA1 IgA2

IgG3

Structure of the Immunoglobulin

Fab

Fc

Structure of the ImmunoglobulinLight Chain

Heavy Chain

Fragment, antigen binding

Fragment crystallizable

region

Domains of the Immunoglobulin

Antibody DomainsV: VariableC: Constant

Fab

Fc

Fragment, antigen binding

Fragment crystallizable

region

Antibodies conjugated with FITC (fluorescein isothiocyanate) and Cy3 (carbocianine 3)

Antihuman anti-FAB antibodyConjugated with

FITC (fluorescein isothiocyanate)

Antihuman antibody conjugated with Cy3 (carbocianine 3)

Myocardium with deposition of Anticardiac antibodies being identified by the antihuman anti-fab FITC antibody

Myocardium with deposition of Anticardiac antibodies being identified by the

antihuman anti-fab FITC antibodies

Anticardiac Antibody

Antihuman anti-fab FITCantibody

Myocardium with no deposition of Anticadiac Antibodies

Now What?

Healthy Myocardium

Heart Failure

Deposition of Anticardiac Autoantibodies in the myocardium identified by the antihuman anti-fab FITC labeled antibodies

Deposition of Anticardiac Autoantibodies in the myocardium identified by the antihuman anti-fab FITC

labeled antibodies

Antibodies

Mitochondria

M7ANT

BCKD-E2

Sarcolemma / Contractile

Proteins

TroponinActin

Myosin

MuscarinicReceptors

B1-AdrenérgicReceptors

Prevalence of Anticardiac IgG antibodies

Samples of healthy

hearts ( 40 pts) without

anticardiac antibody deposition

Patients (71/100 )

with deposition

of anticardiacautoantibo

diesOut of the 71/100

patients with

deposition of

anticardiac antibodies68% was of

the IgG3 subclass

Myocardium of a patient with heart failure and double stain for IgG3 y C3c.

Deposition of FITC labeled antibody directed agains C3c

Deposition of CY3 labeled antibody directed agains IgG3

Overlap of C3C and IgG3 staining

Magnification

• Group A shows a subset of patients who were positive for both IgG3 and for the complement activation marker C3c.

• Group B shows a subset of patients who tested negative for both IgG3and C3c.

• Average Years of disease in patients who tested positive was 11 years to 6 years of those who were negative.

• This suggests that with increasing duration of the disease increases the presence and activation of the immune system

Heart Failure duration (yrs) and activation of the immune system

ConclusionsWe demonstrate for the first time that 70% of patients with heart failure regardless of etiology has deposition of anticardiac autoantibodies

The IgG3 subtype the most commonIt has the largest cell cytotoxic effectActivates complement in the most efficient

It has the potential to cause severe proinflammatory effects.

As disease duration increases so does the presence and activation of the immune system.

Additional studies are needed to understand the role of different classes of antibodies in myocardial dysfunction

These findings support the theory that the immune system plays an important role in the development and progression of cardiac dysfunction and therefore is a reasonable target for future therapeutic modalities.

Thank You

Chassadk@utmb.edu

Christian Assad-Kottner

Christianassad