Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Summary

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Abstract

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults¹. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease^2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption^3-5. A variety of specific antigens have already been identified^2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial^6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals^8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works^10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca²⁺) contents¹². In the last step, the effect of patient IgG on the cell shortening and Ca²⁺ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

Protocol

1. IgG Isolation from Patient Samples

1. Prepare mini-immunoadsorption columns by filling 3 ml anti-IgG sepharose per 2 ml patient EDTA plasma into an empty Econo Pac column. IgG isolation requires at least 2 ml of patient plasma.
2. Place a filter on top of the anti-IgG sepharose, cut open the lower tip of the column and press the filter to reach a flow rate of approximately 1 drop/sec. Let the preservation solution run out of the column.
3. Wash the column with 3 volumes 0.9% NaCl per volume of plasma.
4. Pipette the plasma on the column. When the plasma has completely immersed in the anti-IgG sepharose, wash with the same volume 0.9% N....

Results

Two examples for the measurement of cellular inotropy in field stimulated adult cardiomyocytes (Figure 2) using a myocyte Ca²⁺ and contractility recording system are given below. Figure 3 gives an impression of a control measurement, while in Figure 4 the effect of cardiodepressive antibodies of a patient with DCM is shown.

In both examples, initial cell shortening and Ca²⁺ transient during superfusion with EB exhibi.......

Discussion

The presented method offers a suitable way to detect functionally effective cardiac autoantibodies in patients with DCM of unclear origin. In comparison to other methods, e.g. the detection of functional active antibodies against the β1-adrenoceptor by their impact on cAMP levels⁶, our method is independent of a specific epitope. Of course there are other epitope independent assays described in the literature, i.e. counting the beating rate of neonatal cardiomyocytes¹³, .......

Materials

Name	Company	Catalog Number	Comments
Name of the reagent/equipment	Company	Catalogue number	Comments
Immunosorba preservation solution	Fresenius Medical Care	903609211
Collagenase type 2	Cell System	LS004176
BSA, fatty acid free	Sigma-Aldrich	A6003
Laminin	Sigma-Aldrich	L2020
Fura-2 AM	Sigma-Aldrich	F0888	1 mg/ml in DMSO
Econo Pac Chromatography Columns	BioRad	732-1010
Spectra/Por Biotech Cellulose Ester Dialysis Membrane	Spectrumlabs	131417
4 well Chambered Coverglass	Nunc	155383
Myocyte Calcium and Contractility Recording System	IonOptix
IonWizard 6.0 analysis software	IonOptix