Autoimmunity against cardiac troponin I in ... - Wiley Online Library

European Journal of Heart Failure (2011) 13, 1052–1059 doi:10.1093/eurjhf/hfr098

Autoimmunity against cardiac troponin I in ischaemia reperfusion injury H. Christian Volz1†, Sebastian J. Buss 1†, Jin Li1, Stefan Go¨ser 1, Martin Andrassy 1, ¨ ttl 1, Gabriele Pfitzer 2, Hugo A. Katus 1, and Ziya Kaya 1* Renate O 1 Department of Cardiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany; and 2Institute of Vegetative Physiology, Medical Faculty of Cologne, Germany

Received 15 February 2011; revised 30 April 2011; accepted 13 May 2011; online publish-ahead-of-print 4 August 2011

Aims

Autoimmunity against cardiac troponin I (cTnI) has deleterious effects on the infarcted myocardium early after onset of ischaemia. Here, we explored the impact of cTnI-autoimmunity in the long term. Furthermore, we studied the effects of cTnI-autoimmunity on the infarcted myocardium following revascularization measures in terms of ischaemia reperfusion injury (IRI), which resembles clinical reality more closely. ..................................................................................................................................................................................... Methods After immunization with either cTnI (n ¼ 10) or a control buffer (n ¼ 14), A/J mice underwent chronic coronary artery ligation. Another group of mice immunized with cTnI (n ¼ 13) underwent temporary coronary artery occluand results sion and were compared with non-immunized controls (n ¼ 17). Left ventricular function was evaluated by echocardiography. Hearts were obtained for histological evaluation. Immunological responses were quantified by analysis of cytokine and chemokine patterns as well as anti-cTnI antibody titres. Myocardial inflammation and cardiac dysfunction were detectable as late as 180 days after myocardial infarction (MI). Previous cTnI-immunization enhanced myocardial inflammation and dysfunction. Mice subjected to cTnI-immunization before IRI exhibited a higher inflammation score, an upregulated expression of pro-inflammatory chemokines (IP-10, MIP-1, Ltn, RANTES, TCA-3) and chemokine receptors (CCR2, CCR5), increased interleukin (IL)-2, interferon (IFN)-g, and decreased IL-10 production along with a markedly reduced fractional shortening after IRI compared with the controls. ..................................................................................................................................................................................... Conclusion Our results demonstrate for the first time that cTnI-induced autoimmune response not only leads to increased myocardial inflammation and impaired cardiac function 180 days after chronic coronary artery ligation, but also exacerbates ischaemia/reperfusion injury compared with non-immunized controls. Hence, the presence of cTnIautoimmunity could render subjects more vulnerable to prospective myocardial injury, be it MI, or secondary revascularization measures.

----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords

Ischaemia † Reperfusion injury † Myocardial infarction † Cardiac troponin † Autoimmunity

Introduction The immune response directed against cardiac antigens may be linked to contractile dysfunction in a variety of cardiomyopathies. Cardiac troponin I (cTnI) released into the systemic circulation induces an autoimmune response of both the humoral and cellular immune system, further contributing to myocardial injury. Autoantibodies against cTnI not only have a direct deteriorating influence on cardiomyocytes, as stated by Okazaki et al.,1 but we recently demonstrated that together with the cellular immune †

system, they can induce an extensive inflammatory reaction within the myocardium of mice.2 In the context of myocardial infarction (MI) secondary to chronic coronary ligation, cTnI-induced autoimmunity increases myocardial inflammation and infarct size during the early phase (21 days post-MI).2 Based on these findings, we have now explored the long-term effects of cTnI-autoimmunity on non-revascularized myocardium. However, the mainstay in the clinical management of MI nowadays includes revascularization of the occluded coronary artery. Restoration of coronary blood flow aggravates myocardial

These authors contributed equally to this work.

* Corresponding author. Tel: +49 6221 56 39617, Fax: +49 6221 752885, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2011. For permissions please email: [email protected].

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inflammation, also known as ischaemia reperfusion injury (IRI). Extensive research on the mediators in early IRI identified reactive oxygen species; the complement system, cytokines, and leukocyte accumulation as contributing factors to inflammatory damage after IRI.3 – 7 A number of patients develop cardiac dysfunction despite patent coronary arteries. However, little is known about the immunological effects occurring during the late stage of IRI. We hypothesized that patients presenting with a cTnI autoimmunity and myocardial ischaemia/reperfusion might be at higher risk of enhanced myocardial IRI than non-immunized controls. We, therefore, investigated the involvement of autoimmunity against cTnI in the setting of IRI. Echocardiography was performed to assess cardiac function, while histological specimens and cytokine secretion patterns were evaluated to determine the inflammatory response.

incised in order to expose the heart. After preparation of the left anterior descending (LAD) coronary artery, an 8-0 silk suture was tied around the vessel for ligation. In animals undergoing IRI, loosening of the suture knot re-established coronary blood flow after 30 min of ischaemia.7,8 The chest wall was subsequently closed with a 6-0 Ticron suture. Sham-operated mice underwent the identical procedure without the LAD ligature.

Echocardiography Transthoracic echocardiography was performed with an ATL 5000 echocardiography machine on the day of euthanization as described previously.9 Mice were examined using a dynamic focused 10 MHz probe. The M-mode measurements of the left ventricular parameters were averaged from three cycles. Imaging was done by a physician blinded to the group allocation of the respective mice.

Histopathology and immunohistochemistry

Methods Study animals Female A/J mice (4 – 5 weeks of age, Harlan Winkelmann GmbH, Borchen, Germany) were used in our experiments. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

Experimental protocol in chronic coronary artery ligation Three groups of A/J mice were formed: one group was immunized twice with 150 mg of recombinant cTnI (obtained as described previously2) and 21 days later chronic ligation of the left coronary artery was performed (n ¼ 10). A second group was pre-immunized with control buffer prior to surgery to induce MI (n ¼ 14), whereas a further seven mice served as control group, and thus were preimmunized with control buffer before undergoing a sham operation. On days 21 and 180, echocardiography was performed and blood samples were collected. After 180 days, all mice were sacrificed. The hearts and spleens were then collected according to the protocol published by Goser et al.2 for further analysis.

Experimental protocol in ischaemia/ reperfusion injury Two groups of mice were immunized twice (21 and 14 days prior to the IRI procedure) either with murine cTnI (n ¼ 13, 100 mL emulsion of 150 mg of cTnI per mouse, subcutaneously) or a control buffer (n ¼ 17, 100 mL emulsion of control buffer). Twenty-one days later, IRI was induced in 26 A/J mice via a transient coronary artery occlusion for 30 min and subsequent reperfusion, while four mice (pre-immunized with control buffer) underwent a sham-IRI procedure. Ninety days after the ischaemic event, echocardiography was performed and blood samples collected. Hearts were obtained for histological evaluation and for chemokine expression analysis. Spleens were isolated to profile the cTnI-specific cytokine activation pattern.

Murine model of chronic coronary artery ligation and ischaemia/reperfusion injury After rapid induction of anaesthesia, mice were anaesthetized with isoflurane and intubated according to standard practice. Buprenorphine (0.1 mg/kg) was applied subcutaneously subsequent to intubation. Under direct microscopic visualization, the precordial chest was then

Serial cross-sections of 5 mm thickness each through the entire heart were prepared and stained with haematoxylin and eosin (H&E) in order to define the level of inflammation. Masson’s trichrome staining was used to determine the extent of collagen deposition. An independent examiner blinded to the treatment arm of the respective specimens explored every fifth cross-section and classified them according to the six-tier scoring system published previously2: grade 0 (no inflammation), grade 1 (cardiac infiltration in up to 5% of the preparation), grade 2 (6 – 10%), grade 3 (11 – 30%), grade 4 (31 – 50%), and grade 5 (.50%).

Real-time reverse transcriptase polymerase chain reaction of chemokines and their receptors mRNA was extracted from myocardial tissue and cells using RNAeasy columns (Qiagen). Analysis was performed with iScript cDNA Synthesis Kit (BioRad) according to the manufacturer’s instructions using SsoFast EvaGreen Supermix (BioRad) for amplification. mRNA for IP-10, MCP-1, MIP-1a, MIP-1b, Ltn, RANTES, TCA-3, CCR1, CCR2, and CCR5 were quantified in duplicate. The following primer sequences were applied: mouse IP-10 forward: 5’-GTGTGTGCGTGGCTTCACT-3’, and mouse IP-10 reverse: 5’-GAGATCATTGCCACGATGAA-3’; mouse MCP-1 forward: 5’-CATCCACGTGTTGGCTCA-3’, and mouse MCP-1 reverse: 5’-GATCATCTTGCTGGTGAATGAGT-3’; mouse MIP-1a forward: 5’-CCCAGCCAGGTGTCATTT-3’, and mouse MIP-1a reverse: 5’-CTGCCTCCAAGACTCTCAGG-3’; mouse MIP-1b forward: 5’-CTTCTGTGCTCCAGGGTTCT-3’; and mouse MIP-1b reverse: 5’-TGCCGGGAGGTGTAAGAG-3’; mouse Ltn forward: 5’-GAGACTTCTCCTCCTGACTTTCC-3’, and mouse Ltn reverse: 5’-GGACTTCAGTCCCCACACC-3’; mouse RANTES forward: 5’-GTGCCCACGTCAAGGAGTAT-3’, and mouse RANTES reverse: 5’-TCCTTCGAGTGACAAACACG-3’; mouse TCA-3 forward: 5’-CCCCTGAAGTTTATCCAGTGTTA-3’, and mouse TCA-3 reverse: 5’- GCAGCTTTCTCTACCTTTGTTCA-3’; mouse CCR1 forward: 5’-TGTGTGGACAAAATACTCTGGAA-3’, and mouse CCR1 reverse: 5’- GTGGGGTAGGCTTCTGAA-3’; mouse CCR2 forward: 5’-ACCTGTAAATGCCATGCAAGT-3’, and mouse CCR2 reverse: 5’-TGTCTTCCATTTCCTTTGATTTG-3’; mouse CCR5 forward: 5’-GAGACATCCGTTCCCCCTAC-3’, and mouse CCR5 reverse: 5’-GTCGGAACTGACCCTTGAAA-3’. Thermal cycle was programmed according to the following protocol: 30 s at 958C; 5 s at 958C; and 10 s at 608C. The ribosomal housekeeping gene L32 served as reference. Data were analysed on the basis of the relative expression method (△△Ct method).

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Autoantibodies against cardiac troponin I enzyme-linked immunosorbent assay An enzyme-linked immunosorbent assay (ELISA) was established to measure the titre of autoantibodies against cTnI.2 The 96-well plates were coated overnight at 48C with 5 mg/mL cTnI (100 mL/well) dissolved in bicarbonate buffer (0.1 M NaHCO3/34 mM Na2CO3, pH 9.5). 1×PBS/0.05% Tween 20 served as washing buffer. Plates were then coated with 1% Gelatine (Cold Water Fish, Sigma, 300 mL/ well). After an incubation period of 2 h at 378C and rinsing, IgG (Sigma A2554) diluted to 1:5000 was applied for detection (1 h at room temperature, 100 mg/well). Dilution series of serum samples were performed as follows: 1:100, 1:400, 1:1600, 1:6400, and 1:25 600. Blue Star HRP substrate solution (Diarect) was then applied for 30 min at room temperature (100 mL/well) and the colour reaction was stopped with 0.3 M H2SO4. All samples were measured in duplicate. Optical densities of each well were determined using a microplate reader set to 450 nm. The antibody endpoint titre of each mouse was determined as the highest positive dilution of antibody.

Cardiac troponin I-specific cytokine enzyme-linked immunosorbent assay Isolated splenocytes were cultured (5 × 106 cells/mL) in RPMI 1640 complete medium in a humidified incubator at 378C and 5% CO2. Stimulation was done with cTnI at a concentration of 30 mg/mL. The medium served as negative control, whereas Concanavalin A (Sigma C5275) was used for the positive control. After 48 h of incubation, the supernatants were collected and stored at 2208C. Measurement

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of interleukin (IL)-2, IL-10, and interferon (IFN)-g in the supernatants was performed with the Quantikine cytokine ELISA kits (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to the manufacturer’s instructions.

Statistical analysis For ordinal data the Kruskal – Wallis test was performed followed by the unpaired Mann– Whitney U test for individual comparisons between the sums of ranks. The null hypothesis was rejected when P , 0.05. All calculations were carried out with the statistical software SPSS, version 17.0.

Results Long-term effects of chronic coronary artery ligation in cTnI-pre-immunized mice In order to quantify cardiac performance, we measured fractional shortening (FS) immediately before, and then 21 and 180 days after chronic coronary artery ligation. Control animals revealed a mean FS of 44.6 + 6.5, 42.8 + 5.6, and 41.2 + 3.1%, respectively (not significant, Figure 1). After MI, mice showed a significantly reduced mean FS when pre-immunized with cTnI (FS 25.6 + 1.7% on day 21 and 15.4 + 6.9% on day 180) compared with the MI group without cTnI-pre-immunization (FS 33.6 + 2.0% on day 21 and 25.3 + 4.7% on day 180, P , 0.01) and the sham-operated controls (P , 0.001, Figure 1).

Figure 1 Echocardiographic fractional shortening of mice pre-immunized with cardiac troponin I and undergoing a myocardial infarction (cTnI + MI) compared with mice with a myocardial infarction (buffer + MI) and the sham-operated control group (sham) on days 21 and 180, respectively. Representative histological slides (haematoxylin and eosin on the left, Masson’s trichrome on the right, magnification ×10, day 180) are depicted next to the graph: sham (A – B), buffer + MI (C – D), and cTnI + MI (E – F).10

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Staining with H&E revealed prominent inflammation in cTnI-pre-immunized mice that underwent MI [histoscore 3.0 interquartile range (IQR) 2.1] in contrast to mice without cTnI-pre-immunization (histoscore 1.0 IQR 0.4, P ¼ 0.002). Accordingly, the extent of fibrosis in cTnI-pre-immunized mice with MI (histoscore 3.8 IQR 0.9) was more severe compared with mice that underwent MI alone (histoscore 2.5 IQR 1.4, P ¼ 0.003) as evidenced by Masson’s trichrome staining. In contrast, sham-operated mice showed neither significant inflammation nor increased collagen deposition (histoscore 0) (Figure 1). All mice immunized with cTnI had an anti-cTnI antibody titre of ≥1:25 600. No antibody titre was detectable in animals receiving the control buffer. Mice that underwent MI showed a mean anti-cTnI titre of 1:500. Cardiac troponin I-specific production of IL-2 and IFN-g by splenocytes was higher in cTnI-pre-immunized mice that underwent MI compared with the MI control group (P , 0.05), while there was no significant difference in cTnI-specific production of IL-10 (data not shown).

Effects of ischaemia/reperfusion injury in cTnI-pre-immunized mice Impaired cardiac function after ischaemia/reperfusion injury and cTnI-immunization Mice subjected to cTnI-pre-immunization before IRI showed reduced FS (FS 31.5 + 5.8%) after 90 days. In contrast, FS in mice immunized with control buffer before IRI (FS 37.8 + 1.1%) was significantly higher compared with the latter (P , 0.01, Figure 2). Sham operation did not result in any significant changes in cardiac function compared with untreated controls (FS 44.9 + 2.1 vs. 44.1 + 5.1%, respectively).

Cellular infiltrates and fibrosis after ischaemia/reperfusion injury and cTnI-immunization Haematoxylin and eosin staining of myocardial sections demonstrated a higher histoscore in immunized mice compared with the control group (histoscore 1.5 IQR 2.0 vs. 0.5 IQR 0.5, P ¼ 0.022), while sham-operated mice elicited no signs of inflammation within the myocardium (histoscore 0, Figure 3A). Results from histological specimens stained with Masson’s trichrome were in concordance with these findings: pronounced fibrosis could be found in hearts of cTnI-pre-immunized mice in contrast to the control group (histoscore 1.5 IQR 1.0 vs. 1.0 IQR 0.5, P ¼ 0.057); sham-operated mice showed no significant fibrosis (histoscore 0, Figure 3B).

Innate immune response to ischaemia/ reperfusion injury and cTnI-immunization Chemokines play a pivotal role in myocardial reperfusion injuries.4 mRNA expression of pro-inflammatory cytokines such as IP-10, lymphotaxin (Ltn), regulated upon activation normal T-cell expressed and secreted (RANTES) and T-cell activation gene 3 (TCA-3) was more pronounced in mice that underwent IRI and pre-immunization with cTnI compared with mice that underwent IRI alone (Figure 4A). In concordance with these data, the expression of chemokine receptors CCR2 and CCR5 was significantly upregulated in mice pre-immunized with cTnI before IRI (Figure 4A).

Autoantibody and cytokine production in response to ischaemia/reperfusion injury and cTnI-immunization Subcutaneous injections of cTnI yielded anti-cTnI antibody titres of ≥1:25 600, thereby confirming successful cTnI-immunization. In the control animals no significant antibody titre was detectable (data not shown). Cardiac troponin I-stimulated splenocytes from mice pre-immunized with cTnI showed increased levels of IL-2 (P ¼ 0.016) and IFN-g (P ¼ 0.016) and reduced levels of the anti-inflammatory cytokine IL-10 (P ¼ 0.032) compared with mice who did not undergo cTnI-immunization before the IRI (Figure 4B).

Discussion

Figure 2 Echocardiographic fractional shortening of mice subjected to either cardiac troponin I-pre-immunization + ischaemia/reperfusion injury (cTnI + IRI), control buffer + IRI (buffer + IRI), or control buffer + sham-operation (sham); day 0: initial fractional shortening before any intervention. Squares represent the mean fractional shortening for each group; bands mark the respective standard deviation.

The immune system plays a crucial role in myocardial injury as well as in the healing process depending on the prevailing constellation of inflammatory factors. Following myocardial damage, cardiac proteins including cTnI are released into the systemic circulation. Cardiac troponin I has been shown to correlate with increased mortality in heart failure.11 If mistaken by the immune system as antigen instead of self-protein, these molecules can eventually initiate an autoimmune response.1,2 Accordingly, clinical studies have found anti-cTnI autoantibodies in patients after an acute coronary syndrome.12 There is growing evidence to support the hypothesis that these antibodies interact with membranous cTnI, thereby potentially impairing cardiac function.1 We have previously

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Figure 3 (A) Haematoxylin and eosin staining. Chart representing median histoscores for mice undergoing either cardiac troponin I-pre-immunization + ischaemia/reperfusion injury (cTnI + IRI), control buffer + IRI (buffer + IRI), or control buffer + sham-operation (sham), respectively. Squares represent the median histoscore for each group, bands mark the interquartile range. Representative histological slides (magnification ×10) are depicted next to the graph: sham (A), buffer + IRI (B), and cTnI + IRI (C). (B) Masson’s trichrome staining. Chart representing median histoscores for mice undergoing either cardiac troponin I-pre-immunization + ischaemia/reperfusion injury (cTnI + IRI), control buffer + IRI (buffer + IRI), or control buffer + sham-operation (sham), respectively. Squares represent the median histoscore for each group, bands mark the interquartile range. Representative histological slides (magnification ×10) are depicted next to the graph: sham (A), buffer + IRI (B), and cTnI + IRI (C).

demonstrated that cTnI-autoimmunity exacerbates myocardial inflammation and infarct size 21 days after non-reperfused MI.2 In the present study, we investigated the long-term effects of

cTnI-autoimmunity on the myocardium after non-reperfused MI. Here, we showed that myocardial inflammation and fibrosis as well as functional impairment were prominent as late as


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Figure 4 (A) Real-time reverse transcriptase-polymerase chain reaction of chemoattractants (IP-10, MCP-1, MIP-1a, MIP-1b, Ltn, RANTES, TCA-3) and chemokine receptors (CCR1, CCR2, CCR5) following incubation of splenocytes with cardiac troponin I. Graphs show the fold change of mRNA compared with the untreated sham operated control group. Bands mark the respective standard deviation. *P , 0.05; **P , 0.01; n.s., not significant. (B) Bar charts showing cytokine secretion patterns derived from splenocytes incubated with cardiac troponin I in mice undergoing either cardiac troponin I-pre-immunization + ischaemia/reperfusion injury (cTnI + IRI) or control buffer + IRI (buffer + IRI). Bands mark the respective standard deviation. *P , 0.05.

6 months after MI and even more in the presence of cTnI-induced autoimmunity (Figure 1). Moving closer to clinical reality, we focused on the effects of cTnI-autoimmunity on the myocardium following revascularization measures. Tissue injury caused by ischaemia and subsequent reperfusion is a common pathology encountered in the clinical setting,

especially in the era of percutaneous coronary intervention (PCI). The current concept of early reperfusion in MI improves outcomes by limiting infarct expansion and promoting tissue repair mechanisms.8 However, on the other hand, the re-establishment of coronary blood flow can, paradoxically, also precipitate myocardial injury.3,8,13 Upon restoration of coronary

1058 blood flow, the innate immune system launches a cascade of inflammatory reactions which contribute to parenchymal damage.3 – 6 In our present experimental model, we demonstrate for the first time that both the innate and the adaptive immune response to cTnI are involved during the late stages of IRI. Here, we have shown that an inflammatory reaction was evident up to 90 days after IRI. Subsequent to IRI, different pro-inflammatory chemokines were released and even more in the presence of cTnI-autoimmunity. IP-10 and MCP-1 are thought to be the main factors in the early advent of IRI.4 Beyond their chemotactic properties, they regulate angiogenesis in the healing infarct.4 MCP-1 was not significantly increased, but IP-10 mRNA expression tended to be more pronounced in mice with previous immunization against cTnI before IRI (Figure 4B). Ltn, RANTES, and TCA-3 have been less intensively characterized in the context of early IRI.4 Here, we showed that these chemoattractants were substantially overexpressed 3 months after IRI and even more following cTnI-immunization, suggesting them as mediators of inflammation in the late stages of IRI. Consistent with these findings, the corresponding chemokine receptors CCR2 and CCR5 were upregulated compared with the control group (Figure 4B). As to the adaptive immune response, successful immunization against cTnI was confirmed by high anti-cTnI autoantibody titres. Interestingly, some mice that underwent an MI alone showed low levels of autoantibody titres, whereas mice that underwent an IRI alone did not show any significant anti-cTnI autoantibody production, probably due to the different kinetics depending on the experimental setting. When analysing the cTnI-specific cytokine pattern, we found that the cytokine profile (increased cTnI-specific IL-2 and IFN-g production by splenocytes from cTnI-immunized mice undergoing IRI) pointed towards Th1 polarization, the archetypical inducer of organ specific autoimmunity.14 As an endogenous anti-inflammatory cytokine, IL-10 is paramount in diminishing myocardial injury after IRI, suppressing tissue infiltration of granulocytes and maintaining vessel patency in the reperfused heart.15 In our present study, 90 days after IRI, IL-10 expression was significantly lower in cTnI-immunized mice subjected to IRI compared with the control group. Previous studies have verified the involvement of the immune system in ischaemia/reperfusion injury as the key regulator of the equilibrium between myocardial injury and the healing process of the heart.3 – 6 Which mechanism prevails, largely depends on the predominating inflammatory factors in the particular pathological and/or interventional setting. In a previous report, we demonstrated that cTnI-autoimmunity impairs the myocardium in the early advent of non-reperfused MI. We now demonstrate that these deleterious effects endure beyond this early period, up to 180 days post-MI. Furthermore, we show that inducing an autoimmune response against cTnI exacerbates myocardial inflammation subsequent to ischaemia/reperfusion injury. Our results indicate that a previously induced autoimmune response against cTnI (i.e. exposure to cTnI by any prior cardiac cell injury) predisposes animals to a worse outcome after ischaemia/reperfusion. One limitation of our study is the fact that recombinant cTnI was used in our murine model of myocarditis. Endogenous cTnI has been shown to undergo post-translational glycosylation and acetylation.16,17 Thus, a different immunoreactivity of both forms

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of cTnI (recombinant vs. endogenous) cannot be excluded. A recent study reported that levels of anti-cTnI tend to increase in elderly patients with chronic heart failure following titration of beta-blocker therapy but do not correlate with disease severity.18 Thus, further clinical studies with larger numbers of patients are needed to clarify the pathogenic role of cTnI autoantibodies in patients with ischaemic and dilated cardiomyopathy. Our findings could have a significant impact on the diagnostic and therapeutic approach to the patient with MI. In light of the antagonistic effect of anti-cTnI autoantibodies, their removal from the systemic circulation could be of benefit for these patients, since clinical studies have confirmed an advantage of immunoadsorption therapy in dilated cardiomyopathy.19 – 21 On the other hand, enhancing the cardioprotective effects of IL-10 by exogenous administration have been effective in animal models of IRI.22 Modulating the balance between injury and repair in favour of MCP-1 supports angiogenesis, induces Th2 polarization, which in turn increases IL-10 secretion.4,22 With regard to immunosuppressants, several studies have examined the value of corticosteroids in MI presuming their potentially beneficial effects by limiting the acute inflammatory response of MI.23 However, administration of prednisolone in the context of MI in various experimental animal models has been associated with delayed infarct healing, left ventricular wall thinning, and myocardial rupture.23 To date, 11 clinical trials have been performed in humans, most of them being moderate sized, heterogeneous studies, with differing treatment protocols. None of these studies found conclusive evidence of a detrimental effect of corticosteroids in MI. Moreover, a meta-analysis suggested a possible decrease in mortality with the use of corticosteroids.23 These conflicting results emphasize the need for a better understanding of the inflammatory mechanisms to achieve effective suppression of the injurious processes without impeding myocardial repair. Interestingly, recent clinical trials with cyclosporine show that this may be a promising novel approach to address ischaemia/reperfusion injury.10,24 Applied at the time of PCI, it is conceivable that the immunosuppressant could limit the size of infarction. The ascribed targets of the drug are the mitochondrial permeability transition pores. However, to what extent the involvement of lymphocytes could possibly have contributed to the results remains uncertain. In this regard, more research needs to be done to provide the rationale for the use of these treatment modalities.

Acknowledgements ¨ zay Kaya and Theresa Tretter for critically The authors thank O reading the manuscript.

Funding Deutsche Forschungsgemeinschaft (KA1797/3-1 to Z.K., SFB612 to G.P.) Conflict of interest: none declared.

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