This work was supported by the Sonderforschungsbereich Transregio 19 (SFB/TR19, C2) of the Deutsche Forschungsgemeinschaft (DFG), the Federal Ministry of Economics and Technology project ZIM-KF 2727801MD0 and the Centre for Innovation Competence - Humoral Immune Responses in Cardiovascular Diseases (ZIK-HIKE, BMBF FKZ 03Z2CN12) of the Federal Ministry of Education and Research, Germany. Housing and experiments with animals were performed in accordance with the recommendations of the Society for Laboratory Animal Science (Gesellschaft f&uuml;r Versuchstierkunde, GV-SOLAS) and the Federation of European Laboratory Animal Science Association (FELASA).

1. IgG Isolation from Patient Samples
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Wash the column with 3 volumes 0.9% NaCl per volume of plasma.</li>
	<li>Pipette the plasma on the column. When the plasma has completely immersed in the anti-IgG sepharose, wash with the same volume 0.9% NaCl.</li>
	<li>Pipette one plasma volume of 0.2 M glycine HCl, pH 2.8 on the column and let immerse into the sepharose. Add another volume of glycine HCl onto the column. Collect the flow through in a 15 ml tube and adjust the pH to 7.3 with 0.5 M Tris-HCl, pH 8.0.</li>
	<li>To regenerate the column, wash with 3 plasma volumes glycine HCl. After a final wash with 2 volumes 0.9% NaCl, the column may be used for the next plasma sample. Alternatively, the column can be filled with preservation solution and stored at 4 - 8 &deg;C for up to 4 weeks.</li>
	<li>For use on cardiomyocytes, the collected IgG fraction has to be dialyzed against experimental buffer (EB). For preparation of EB, add 117 mM NaCl, 2.8 mM KCl, 0.6 mM MgCl2, 1.2 mM KH2PO4, 1.2 mM CaCl2,10 mM HEPES and 20 mM glucose to 1 L distilled water on a magnetic stirrer and adjust pH to 7.3.</li>
	<li>Fill the IgG fraction to a cellulose ester dialysis membrane tube with a molecular weight cut off of 100 kDa. Put the tube in 1 L EB and stir slowly with a magnetic stirrer at 4 &deg;C. After 8 hr, transfer the dialysis tube into 3 L of fresh EB and dialyze for another 20 hr at 4 &deg;C.</li>
	<li>Following dialysis, heat the sample for 30 min at 57 &deg;C in a water bath to inactivate complement factors. Determine total IgG concentration and prepare aliquots containing 330 &mu;g IgG each. When adding the sample to cardiomyocytes for measurement (step 4.3), fill up the aliquots to 1 ml with EB. Undiluted aliquots can be stored at -20 &deg;C until further use.</li>
</ol>
2. Isolation of Rat Cardiomyocytes
<ol>
	<li>For preparation of Ca2+-free isolation buffer (IB), add 110 mM NaCl, 2.6 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM HEPES and 11 mM glucose to 1 L distilled water using a magnetic stirrer. Adjust pH to 7.4 at room temperature and filter through a 0.2 &mu;m bottle top filter for sterilization.</li>
	<li>Fill 80 ml of IB in the upper reservoir of a thermostated Langendorff perfusion system. Adjust thermostat settings to maintain a buffer temperature of 37 &deg;C and aerate buffer with 95% O2/5% CO2 for 30 min.</li>
	<li>Weigh approximately 10,000 - 12,000 U of collagenase type II, 175 mg fatty acid free BSA and dissolve in 20 ml IB containing 22.5 &mu;l of a 100 mM CaCl2 stock solution. Control the pH of the isolation buffer in the upper reservoir and adjust if required.</li>
	<li>Anaesthetize a Wistar rat of 175 - 200 g with 375 &mu;g/kg body weight thiopental. Add 2500 IU Heparin to thiopental solution. After thoracotomy, excise the heart carefully with an intact aortic arch and transfer it into ice-cold 0.9% NaCl. Clean the heart from blood and surrounding tissue and transfer to fresh 0.9% NaCl. Expose the aorta, open the flow reducer of the Langendorff system to obtain a dropping speed of 2-3/sec and attach the heart to the cannula.</li>
	<li>Perfuse the heart for up to 3 min with a flow rate of 1 drop/sec to wash out remaining blood. Start to circulate the IB in the Langendorff system. Remove 20 ml buffer from the upper reservoir, fill in the collagenase solution and refill as much buffer as required to reach 80 ml. Circulate the collagenase solution for another 27 min. Flow rate should be controlled periodically and maintained at 1 drop/sec using the flow reducer.</li>
	<li>Detach the heart from the cannula, clean from atria and aorta and chop into small pieces. Allow the collagenase solution to run into the lower reservoir, reduce the volume to a maximum of 40 ml and further digest the chopped ventricles for 10 - 15 min under continuous aeration with 95% O2/5% CO2. Afterwards, filter the cell suspension through a 200 &mu;m mesh size gauze into a 50 ml tube.</li>
	<li>Restore the Ca2+ content of the cells in 3 steps: centrifuge the cell suspension at 43 x g for 2 min at room temperature and resuspend cells in 10 ml IB containing 200 &mu;M CaCl2. Centrifuge again and resuspend cells in 10 ml IB with 500 &mu;M CaCl2. After a final centrifugation resuspend cardiomyocytes in sterile filtered EB (see 1.8) to a density of 50,000 cells/ml.</li>
</ol>
3. Fura-2 Staining of Cardiomyocytes
<ol>
	<li>Coat a 4 well chambered coverglass with 10 &mu;g laminin per well and wait until it is fully dried.</li>
	<li>Fill 1 ml of the cardiomyocyte suspension into each well using a micropipette with a cut tip. Allow the cells to adhere for 1 hr at room temperature.</li>
	<li>Dilute 10 &mu;l of the 1 mg/ml Fura-2 AM stock solution in 5 ml EB. Keep the staining solution in the dark.</li>
	<li>Take off buffer and unattached cells from the coverglass and pipette 0.5 ml of the staining solution into each well. Incubate the cells for 10 min under moderate shaking. Subsequently, take off the staining solution, wash cells once with 1 ml EB and finally fill 0.5 ml EB into each well. Avoid direct light during the staining process and always keep the stained cells in the dark.</li>
</ol>
4. Recording of Cell Shortening and Ca2+ Transients
<ol>
	<li>Place the chambered coverglass on an inverse fluorescence microscope connected to a myocyte calcium and contractility recording system. Position a custom made electrode with an influx and efflux tube in the first well. Superfuse cardiomyocytes with 1 ml/min EB and start the electric stimulation (20 V, 1 Hz, 5 msec duration). Allow the cells to adapt to the stimulation for about 2 min.</li>
	<li>Choose an intact rod shaped cardiomyocyte with clear striation and constant contraction. Position the cell in the center of the visual field and open the camera channel. Orientate the cell horizontally within the video area by rotating the cell framing adapter and moving the microscope stage.</li>
	<li>Adjust the edge detecting control elements of the video area (Figure 2), open the fluorescent channel and record 10 - 15 contractions to obtain the initial cell shortening and Ca2+ transient.</li>
	<li>Close the microscope light channel to avoid bleaching of the fluorescence stain. Switch the flow through from EB to the sample bypass with a 3-way valve and superfuse cardiomyocytes with 1 ml of patient IgG. Stop the pump just before air reaches the well.</li>
	<li>Open the light channel and record another 10 - 15 contractions to obtain the acute cell response. Pause the recording and close the light channel again. Repeat recording after 2 and 5 min.</li>
	<li>Take out the electrode of the well. Clean the tubes thoroughly with EB and continue with the next well of the chambered coverglass.</li>
	<li>Analyze the cell shortening and Ca2+ transient with the IonWizard software using the "Edge-Length/Length" and the "fura 2-Numeric subtracted/Ratio" window, respectively. Calculate the percent change from the initial cell shortening and Ca2+ transient of the peak height referred to the baseline (bl%peak h) for the acute, the 2 min and the 5 min response.</li>
</ol>

<ol>
	<li>Roger, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2011+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2011 update: a report from the American Heart Association.</a> Circulation. 123 (4), e18 (2011).</li><li>Caforio, A. L., Vinci, A., Iliceto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-heart+autoantibodies+in+familial+dilated+cardiomyopathy.">Anti-heart autoantibodies in familial dilated cardiomyopathy.</a> Autoimmunity. 41 (6), 462 (2008).</li><li>Yoshikawa, T., Baba, A., Nagatomo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+mechanisms+underlying+dilated+cardiomyopathy.">Autoimmune mechanisms underlying dilated cardiomyopathy.</a> Circ. J. 73 (4), 602 (2009).</li><li>Felix, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+effects+of+immunoadsorption+and+subsequent+immunoglobulin+substitution+in+dilated+cardiomyopathy:+Three-month+results+from+a+randomized+study.">Hemodynamic effects of immunoadsorption and subsequent immunoglobulin substitution in dilated cardiomyopathy: Three-month results from a randomized study.</a> Journal of the American College of Cardiology. 35 (6), 1590 (2000).</li><li>Staudt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+immunoglobulin+G3+subclass+in+dilated+cardiomyopathy:+results+from+protein+A+immunoadsorption.">Role of immunoglobulin G3 subclass in dilated cardiomyopathy: results from protein A immunoadsorption.</a> Am. Heart J. 150 (4), 729 (2005).</li><li>Nikolaev, V. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+fluorescence+method+for+the+rapid+detection+of+functional+beta1-adrenergic+receptor+autoantibodies+in+heart+failure.">A novel fluorescence method for the rapid detection of functional beta1-adrenergic receptor autoantibodies in heart failure.</a> J. Am. Coll. Cardiol. 50 (5), 423 (2007).</li><li>Jahns, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+activating+human+beta1-adrenergic+receptors+are+associated+with+reduced+cardiac+function+in+chronic+heart+failure.">Autoantibodies activating human beta1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure.</a> Circulation. 99 (5), 649 (1999).</li><li>Gocayne, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+structure+of+rat+cardiac+beta-adrenergic+and+muscarinic+cholinergic+receptors+obtained+by+automated+DNA+sequence+analysis:+further+evidence+for+a+multigene+family.">Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.</a> Proceedings of the National Academy of Sciences. 84 (23), 8296 (1987).</li><li>Jaenicke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+sequence+of+the+human+beta-myosin+heavy+chain+gene+and+a+comparative+analysis+of+its+product.">The complete sequence of the human beta-myosin heavy chain gene and a comparative analysis of its product.</a> Genomics. 8 (2), 194 (1990).</li><li>Piper, H. M., Volz, A., Schwartz, P., Piper, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Culture Techniques in Heart and Vessel Research. , (1990).</li><li>Kubin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+endothelial+cells+remodel+cultured+adult+cardiomyocytes+and+increase+their+survival.">Microvascular endothelial cells remodel cultured adult cardiomyocytes and increase their survival.</a> Am. J. Physiol. 276 (6 Pt. 2), H2179 (1999).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J. Biol. Chem. 260 (6), 3440 (1985).</li><li>Magnusson, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+idiopathic+dilated+cardiomyopathy.+Characterization+of+antibodies+against+the+beta+1-adrenoceptor+with+positive+chronotropic+effect.">Autoimmunity in idiopathic dilated cardiomyopathy. Characterization of antibodies against the beta 1-adrenoceptor with positive chronotropic effect.</a> Circulation. 89 (6), 2760 (1994).</li><li>Wakefield, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+in+vitro+methods+to+safety+pharmacology.">The application of in vitro methods to safety pharmacology.</a> Fundam. Clin. Pharmacol. 16 (3), 209 (2002).</li><li>Kajzar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+physiological+conditions+for+cell+analyses:+forces+of+heart+muscle+cells+suspended+between+elastic+micropillars.">Toward physiological conditions for cell analyses: forces of heart muscle cells suspended between elastic micropillars.</a> Biophys. J. 94 (5), 1854 (2008).</li></ol>

No conflicts of interest declared.

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 &beta;1-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, ...

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

measurement of antibody effects on cellular function of isolated cardiomyocytes

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 disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 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 crucial6,7. 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 mammals8,9. 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 works10,11. 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.

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...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

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.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

Research

JoVE Journal

Immunology and Infection

11.8K Views.  University Medicine Greifswald. 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.