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Cardiovascular Research 1999 44(1):91-100; doi:10.1016/S0008-6363(99)00204-7
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Copyright © 1999, European Society of Cardiology

Disturbance of myocardial energy metabolism in experimental virus myocarditis by antibodies against the adenine nucleotide translocator

Karsten Schulze*, Bernhard Witzenbichler, Claudia Christmann and Heinz-Peter Schultheiss

Department of Cardiology, Benjamin Franklin Hospital, Free University of Berlin, Berlin, Germany

* Corresponding address. Medizinische Klinik II, Kardiologie, Universitätsklinikum Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. Tel.: +49-30-8445-4162; fax: +49-30-8445-4141

Received 3 February 1999; accepted 31 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Objective: The adenine nucleotide translocator (ANT) of the inner mitochondrial membrane is an autoantigen in myocarditis and in dilated cardiomyopathy. Clinical and experimental studies showed that specific autoantibodies inhibit the transmembrane nucleotide transport. In isolated hearts of guinea pigs immunized with the ANT, energy metabolism is disturbed. This metabolic disorder is related to functionally active specific antibodies and to a reduced heart function. This study tests whether similar immunological, metabolical and functional responses also occur in experimental virus myocarditis. Methods and results: Experimental virus myocarditis was induced in A.SW/SnJ-mice by Coxsackie B3 virus infection. Specific antibodies against the ANT were detected by Western Blot in 14 out of 19 infected animals. In the isolated perfused hearts of five of these 14 mice cytosolic and mitochondrial ATP/ADP-ratios, determined by nonaqueous fractionation, were significantly altered, signalling a reduced ANT function [cytosolic ATP/ADP: 59±18 vs. 136±20 (controls), mitochondrial ATP/ADP: 4.2±1.0 vs. 1.1±0.3], all P<0.05. Also, left ventricular pressure [43±9 vs. 78±6 mmHg (noninfected controls)], rate-pressure product (15.8±3.2 vs. 30.5±3.0 mmHg/min/1000), dp/dt (2410±222 vs. 3250±118 mmHg/s), and oxygen consumption (4.7±0.9 vs. 7.3±0.7 µmol/g/min), all P<0.05, were lowered. Conclusion: The data support the hypothesis that a virus infection alters cardiac energy metabolism and function by an antibody-mediated modulation of the function of the ANT.

KEYWORDS Myocarditis; Cardiomyopathy; Energy metabolism; Mitochondria; Hemodynamics


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 
An episode of acute myocarditis can initiate cardiomyopathy and heart failure. Clinical and experimental data support immune mechanisms playing a major role in the pathogenesis of myocarditis and dilated cardiomyopathy [1–3]. Most clinical cases of myocarditis in humans are suspected to be of viral etiology. The mechanisms by which an acute virus infection or chronic virus persistence alter the immune system have not yet been completely disclosed [4,5].

Autoantibodies against a wide spectrum of myocardial antigens are found in human virus myocarditis [3,6–10]. It remains doubtful, however, whether they are all relevant for the development of subsequent heart failure. In previous studies, we identified the adenine nucleotide translocator (ANT) as an autoantigen in patients with myocarditis and dilated cardiomyopathy [10]. The autoantibodies directed against the ANT are organ- and conformation-specific and inhibit the nucleotide exchange in vitro by blocking the substrate-binding site of the carrier protein [11,12]. In isolated beating hearts of guinea pigs, immunized with the ANT protein, specific antibodies were generated, the cytosolic–mitochondrial difference of the phosphorylation potential of ATP was lowered, reflecting a dysfunction of the ANT, and heart function was disturbed correlating with the degree of ANT inactivation [13–15].

These experimental findings argue against the notion that all of the antibodies found in myocarditis and dilated cardiomyopathy are a mere epiphenomenon. Recently, a main autoimmunogenic determinant of the ANT could be identified. Using synthetic peptides, a cross-reaction was shown between homologous amino acid sequences of the ANT and the Coxsackie B3 virus. Thus, molecular mimicry could be relevant for the induction of autoantibodies [16].

Murine models of Coxsackie B3 virus-induced myocarditis have been established. Infection of A-strain mice with this enterovirus induces a histologically well-defined myocarditis with a characteristic time-course that resembles human myocarditis [17,18].

Therefore, it was the first aim of this study to show an autoimmune response against the ANT by infecting A-strain mice with Coxsackie B3 virus. In addition, we wanted to observe whether this leads to changes in cardiac energy metabolism typical for ANT dysfunction, and to a subsequently disturbed cardiac function. Only by detecting specific antibodies plus ANT-inactivation plus cardiac dysfunction in experimental myocarditis can the experimental data on ANT-immunized guinea pigs be linked to the clinical findings, thus further uncovering the mechanism of deterioration of cardiac performance after viral myocarditis.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusion
 References
 
2.1 Infection procedure
Three-weeks-old A.SW/SnJ female mice were purchased from the Jackson Laboratories, Bar Harbour, Maine, USA. Nineteen mice were inoculated intraperitoneally with Coxsackie B3 virus, strain Nancy. Virus at a concentration of 2x10 [4] PFU (plaque forming units) per 200 µl PBS (phosphate buffered saline) buffer was injected. Ten control animals received a virus-free preparation that was diluted in the same manner as the Coxsackie B3 inoculum. The infected animals were housed in microcirculation cages. This protocol conformed 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 1996).

2.2 Western blot
Twenty weeks after infection, blood was taken from infected and control mice by retro-orbital bleeding. The solubilization and isolation of the ANT followed the procedure previously described [11]. The isolated ANT, the total protein from calf heart mitochondria, and marker proteins were separated on sodium dodecyl sulfate (SDS) polyacryamide slab gels using a minigel system (Biometra). The gels were run using a 0.25 M Tris buffer, pH 8.7, containing 30% glycine and 0.5% SDS. Gels were either stained with Coomassie brilliant blue or electroblotted for 20 min with 0.5 amperes onto nitrocellulose sheets, 0.45-mm thick (Bio Rad). The nitrocellulose was placed for 1 h in PBS buffer supplemented with 1% Tween 20 (Merck). Afterwards it was incubated overnight at 4°C with mice sera in a dilution of 1:70. To remove unbound antibodies, several washings were performed with 1% Tween 20 in PBS buffer. The washed nitrocellulose sheets were incubated with rabbit anti-mouse antibodies (Dianova). After another washing the sheets were labelled with 125I protein A (New England Nuclear) for 1 h, dried, and exposed to a Cronex film at –80°C.

2.3 The isolated heart preparation
Twenty weeks after infection the mice were anaesthetized by diethylether, the thorax cavity was opened, and the hearts were arrested by 0.9% ice cold sodium chloride solution. A steel cannula of 0.8-mm inner diameter was fixed into the aorta. Before starting perfusion, hearts were submersed in saline to derive heart weights from the respective volume changes. According to the Langendorff technique the hearts were retrogradely perfused. The non-recirculating Krebs–Henseleit buffer contained (in mM): 127 NaCl, 4.7 KCl, 24.9 NaHCO3, 1.25CaCl2, 0.6 MgSO4 and 1.2 KH2PO4, 0.3 pyruvate, 5.5 glucose, and 5 U/l insulin. The buffer was equilibrated at 37°C with 95% O2 and 5% CO2 (pH 7.4). Perfusion pressure was 80 cm H2O. A latex balloon type IVB20 (Biomedix) was inserted into the left ventricle via the incised left atrium and pressurized to 10 cm H2O. The developed ventricular pressure was continuously recorded by a Statham P23Db strain gauge. From the phasic pressure, signal heart rate and dp/dt were derived by a HRM 669 heart rate module and a type 575 slope quotient coupler (Hugo Sachs Electronics), respectively. The pulmonary artery was cannulated to yield coronary flow and oxygen tension, measured by a clark type electrode (Diamond General). To avoid flow-dependent artifacts, the electrode was placed in a bypass of the coronary effluent in which flow was kept constant at 0.5 ml/min. After 20 min of stabilization, functional data were recorded and the hearts were freeze clamped.

2.4 Nonaqueous fractionation
To avoid further metabolic processes, the freeze-clamped hearts were pulverized in a mortar filled with liquid nitrogen, lyophilized, and stored in heptane/carbon tetrachloride. During lyophilization all formerly dissolved metabolites and enzymes cling to the membrane wall of their respective cellular compartment. After ultrasonic disruption of the cells into small membrane fragments, insufficiently homogenized particles, mainly consisting of connective tissue, were removed by successive filtration through columns filled with glass beads of 1.0 and 0.4-mm diameter. The purified homogenate then was subject to density gradient centrifugation (4 h at 16 000 g). Density gradients (1.29–1.38 g/ml) were produced by continuous variation of the volume ratio of the two constituents heptane (0.69 g/ml) and carbon tetrachloride (1.59 g/ml). Since lyophilized mitochondrial membranes are lighter than cytosolic structures, fractions with differing proportions of cytosolic and mitochondrial proteins can be obtained. Each of the eight fractions per gradient was subdivided into two aliquots. In the first aliquot the total protein content and the activities of the cytosolic and mitochondrial marker enzymes, phosphoglycerate kinase (PGK) and citrate synthase (CS), were determined photometrically. In the second aliquot the contents of ADP, ATP, creatine and creatine phosphate were analyzed using high-performance liquid chromatography (HPLC).

2.5 High-performance liquid chromatography
The reversed-phase ion pair chromatographic method for measuring adenine nucleotides was used as described by Hammer et al. [19]. The Kontron HPLC system consisted of two type 420 pumps, a variable Uvikon 735 LC wavelength detector, a 80286 personal computer for solvent delivery programming, data aquisition and integration, and an autosampler (type 460) with a 20-µl Rheodyne high pressure injector loop. A Hewlett-Packard 5-µm RP-18 column (100x2.1 mm) and a Kontron RP-18 guard cartridge system (15x3.1 mm) were used.

For extraction of metabolites 0.6 N perchloric acid together with calcined quartz sand was given to the metabolite aliquots, that were shaken vigorously with a micro-dismembranator (Braun–Melsungen). The acid extract was neutralized by an ice-cold 4:1 Freon/trioctylamine mixture and centrifuged for 1 min. The clear aqueous layer was removed with a Hamilton pipette and 20 µl were injected immediately into the HPLC system. The gradient system consisted of two buffers at a constant flow-rate of 0.2 ml/min. Buffer A was a solution of 30 mM KH2PO4 and 7.5 mM tetrabutylammoniumphosphate, pH 5.45. Buffer B contained of 50/50 (v/v) acetonitrile, 30 mM KH2PO4, and tetrabutylammoniumphosphate, pH 7.0. All reagents were prepared with HPLC-grade water (Merck), filtered through a 0.2-µm membrane filter (Millipore), and degassed by helium insufflation. The eluent gradient started with 5% buffer B, increasing continuously to 50% buffer B within 20 min. After 6 min of isocratic 50% buffer B, all chromatographic peaks were eluted and the column was re-equilibrated for the next sample with 5% buffer B for 10 min. All chromatogram peaks were identified at 254 nm by comparison with retention times of known standards, by wavelength ratios, and by enzymatic shift studies.

2.6 Calculation of intracellular metabolite concentrations
The known total content of a metabolite (Mtot) in each fraction of the density gradient is the sum of its mitochondrial and cytosolic portions, thus:

Formula (1)
In addition, the activities of the marker enzymes PGK and CS, determined in each fraction, correlate with the membrane content and the metabolite content of the respective cellular compartment. This relation between marker enzyme and compartmental metabolite content is constant in all fractions of the gradient and is expressed as:

Formula (2)

Formula (3)
The combination of the Eqs. (1)–(3)GoGo, and division by the CS-content results in the linear expression:

Formula
The factors a and b can be readily obtained by linear regression, whereupon the cytosolic and mitochondrial metabolite contents can be calculated from Eqs. (2) and (3)Go.To derive subcellular metabolite concentrations from the contents yielded by nonaqueous fractionation and HPLC, intracellular water contents were assumed to be 5.82 µl/mg cytosolic protein and 1.0 µl/mg mitochondrial protein. [20] Cytosolic protein contributes about 40%, and mitochondrial protein about 60% to the total myocardial protein. To obtain free cytosolic ADP concentrations from the equation of the creatine phosphokinase reaction, an equilibrium constant KCPK of 2.04x109 and an intracellular pH of 7.12 (own 31P-NMR data from identically perfused mice hearts) was employed [21].

2.7 Statistical analyses
Student’s t-test was used for the comparison of data from hearts of infected mice versus hearts of the noninfected control group. Values are means±standard deviation (SD). For curve fitting of the hyperbolic regression lines in Figs. 4 and 5Go the Marquardt–Levenberg algorithm was used.


Figure 4
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  		Fig. 4 Relation of cytosolic (upper panel) and cytosolic/mitochondrial (lower panel) ATP/ADP ratios with: the rate–pressure product (mmHg/min/1000)

 

Figure 5
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  		Fig. 5 Relation of cytosolic (upper panel) and cytosolic/mitochondrial (lower panel) ATP/ADP ratios with the dp/dt (mmHg/s) in isolated Langendorff-perfused hearts of Coxsackie B3 virus-infected and noninfected A.SW mice. Diamond symbols: noninfected controls (n=10). White (open) circles : infected, antibody negative (n=5). Grey circles: infected, antibody-positive, normal mitochondrial ATP (n=9). Black circles: infected, antibody-positive, heightened mitochondrial ATP (n=5).

 

   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusion
 References
 
3.1 Generation of ANT-specific antibodies in Coxsackie B3 virus-infected A.SW-mice
Twenty weeks after Coxsackie B3 virus infection, the sera of infected (n=19) and noninfected mice (n=10) were tested for reactivity with mitochondrial antigens by Western blot. The ANT forms a characteristic band at 30 000 daltons among these antigens that were presented by electrophoretic separation of mitochondrial proteins isolated from calf hearts. An antibody binding at the 30 000 dalton band was observed in 14 out of the 19 sera of A.SW/SnJ mice infected with the Coxsackie B3 virus, but in none of the sera from the ten noninfected control mice. Fig. 1 shows a representative blot. Thus, twenty weeks after virus infection {approx}75% of the infected mice had generated specific antibodies against the ANT protein.


Figure 1
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  		Fig. 1 Western blot for the detection of antibodies against the ANT. Tracks A–C: polyacrylamide slab gels stained with Coomassie blue. A: marker proteins (molecular weight in daltons). B: total heart mitochondrial proteins. C: isolated ANT (32 000 daltons). Tracks D–E: nitrocellulose blots, incubated with serum from Coxsackie B3 virus-infected A.SW mice, rabbit anti-mouse antibodies, and labelled with 125I protein A. D: total heart mitochondrial proteins. E: isolated ANT.

 
3.2 Changes in cardiac function due to Coxsackie B3 virus infection
Twenty weeks after virus infection the hearts of all mice were isolated and perfused retrogradely. Hearts of infected animals (174±29 mg) were heavier than those from noninfected control mice (138±19 mg), P<0.05. Routinely, the intactness of the preparation was controlled by correlating myocardial oxygen consumption with the rate–pressure product. A sufficiently high linear correlation coefficient of r=0.94 was calculated.

Heart rate was identical in all groups. Hearts from virus-infected animals that had not generated specific antibodies against the ANT performed like the noninfected controls (Fig. 2). Coronary flow (CF), dp/dt, left ventricular pressure (LVP), myocardial oxygen consumption (MVO2), and the rate-pressure product (RPP) were lowered in hearts from infected antibody-positive mice vs. antibody-negative control mice (P<0.05): CF (ml/min) 1.9±0.3 vs. 2.3±0.3, dp/dt 2780±331 vs. 3250±118, LVP (mmHg) 58±13 vs. 78±6, MVO2 (µmol/g/min) 5,8±1.0 vs. 7.3±0.7, RPP (mmHg/min/1000) 21.4±5.1 vs. 30.5±3.0. When dividing the antibody-positive group into two subgroups: (A) no metabolic alterations, and (B) with metabolic alterations (see below) it shows that the changes of heart function were nearly exclusively due to marked alterations in subgroup B, whereas heart function in subgroup A did not differ from noninfected control mice (Fig. 2). Relative myocardial oxygen consumption is higher in hearts of subgroup B if compared to LVP and the rate-pressure product: (MVO2/LVP [µmol/min/g/mmHgx103]: 111.1±12.3 vs. 93.7±6.4 (controls), P<0.05; MVO2/RPP [µmol/g/mmHgx106]: 302±28 vs. 240±13 (controls), P<0.05.


Figure 2
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  		Fig. 2 Functional parameters of isolated Langendorff-perfused hearts of A.SW mice. Columns show (from left to right): control hearts from noninfected mice (white, n=10), hearts from infected mice not having generated antibodies against the ANT (light grey, n=5), hearts from infected mice with specific antibodies against the ANT and normal mitochondrial ATP concentration (dark grey, n=9), hearts from infected mice with specific antibodies against the ANT and mitochondrial ATP concentration above the mean±2SD level of the controls (black, n=5). Error bars represent standard deviation. P<0.05 versus the control group is marked by an asterisk (*).

 
3.3 Changes in energy metabolism after Coxsackie B3 virus infection
Total myocardial contents of ADP, ATP, creatine, and creatine phosphate (CP) were measured in an unfractionated portion of myocardial tissue-homogenate (Table 1). Respective cytosolic and mitochondrial concentrations (mmol/l) were measured by nonaqueous fractionation (Table 2). Measured and back-calculated metabolite contents (from concentration data) did correspond (see Table 1), indicating that no substrates or enzymatic activity was lost during fractionation.


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  		Table 1 Metabolite contents in isolated perfused hearts from A.SW mice infected with Coxsackie-B3 virus

 

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  		Table 2 Metabolite concentrations in isolated perfused hearts from A.SW mice infected with Coxsackie-B3 virus

 
All data from infected, but antibody-negative mice were within the noninfected control range. Hearts from infected and antibody-positive mice showed the following changes (all P<0.05 vs. controls): (1) Total content of creatine phosphate (CP) was lowered, ADP and creatine were heightened (Table 1, bottom row). (2) Subcellular concentrations were heightened for cytosolic ADP+creatine and mitochondrial ATP+CP, and lowered for cytosolic CP (for mitochondrial ADP and cytosolic ATP nonsignificantly) (Table 2, bottom row). Within the antibody-positive group a subset of five hearts shows a mitochondrial ATP concentration far above the mean±2SD level of the control group (11.7±1.8 vs. 5.1±1.0 mM). In this subcollective, not only the observed shifts in total metabolite contents and subcellular metabolite concentrations were strongly pronounced (see Tables 1 and 2Go), but also heart function was markedly reduced (see Fig. 2).

3.4 ATP/ADP ratios and heart function
ATP/ADP were lowered in the cytosol of infected and antibody-positive hearts by 37%: 85±27 vs. 136±20 (controls), P<0.05, and were doubled in the mitochondria: 2.3±1.6 vs. 1.1±0.3, P<0.05 (see Fig. 3). ATP/ADP ratios of the subgroup of hearts with pronounced increases in mitochondrial ATP were 59±18 in the cytosol and 4.2±1.0 in the mitochondria, both significantly different from the control group. Cardiac function and oxygen consumption correlated with cytosolic ATP/ADP ratios and with the quotient of the cytosolic/mitochondrial ATP/ADP ratio, a measure of the functional activity of the ANT, as shown for dp/dt (Fig. 4) and the rate–pressure product (Fig. 5).


Figure 3
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  		Fig. 3 Cytosolic (left panel) and mitochondrial (right panel) ATP/ADP ratios in isolated perfused hearts of A.SW mice, assessed by nonaqueous fractionation. The left pair of columns represent the hearts of antibody-negative mice: white (left)=controls, noninfected (n=10), light-grey (right)=infected (n=5). The right pair of columns represent the hearts of mice with specific antibodies against the ANT: dark grey (left)=total group (n=14), black (right)=subgroup with elevated mitochondrial ATP concentrations (>mean+2SD of controls) (n=5). Error bars represent standard deviation. P<0.05 versus the control group is marked by an asterisk (*).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusion
 References
 
The adenine nucleotide translocator (ANT) of the inner mitochondrial membrane is a well characterized autoantigen in myocarditis and dilated cardiomyopathy. Organ- and conformation-specific autoantibodies were shown in patients with these diseases [10]. Clinical findings give evidence that antibodies against the ANT or the carrier itself play a pathophysiologic role: First, sera of patients with myocarditis and dilated cardiomyopathy inhibit the nucleotide exchange on isolated mitochondria [22]. Second, carrier function in isolated mitochondria of explanted hearts from patients with dilated cardiomyopathy is reduced [23]. Third, carrier concentration is elevated in endomyocardial biopsy samples and explanted hearts of patients with cardiomyopathy, possibly to compensate for antibody-mediated inactivation of carrier molecules [24,25]. Forth, the presence of carrier-inactivating antibodies in the sera of patients with myocarditis determines the prognosis of the disease [26].

The infection with a cardiotropic virus, mostly of the enterovirus type, may initiate myocarditis and cardiomyopathy in genetically predetermined patients [27–29]. Susceptible H-2 oncogenic mice strains are an established animal model for studying human myocarditis. The A.SW strain is known to respond to an enterovirus inoculation with a well characterized myocarditis that histologically resembles the human disease. A heart specific autoimmune response after Coxsackie virus infection has namely been shown in this strain [18].

Based on the possible link between viral infection and induction of autoimmunity we first investigated in this study whether an immune reaction against the ANT can be initiated by Coxsackie-B3 virus infection in A.SW mice: In 74% (14 of 19 sera) Western blot analyses detected specific antibodies against the ANT protein. This observation shows that the ANT is of antigenic potency also in murine virus myocarditis and affirms the clinical observations of the occurrence of antibodies against the ANT in sera from patients with virus myocarditis.

To further assess whether the Coxsackie virus infection induces a relevant disturbance of ANT-function it is necessary to detect specific alterations in cellular energy metabolism. An established parameter of ANT-function is the cytosolic–mitochondrial difference of the phosphorylation potential of ATP [30]. As a working measure the cytosolic and mitochondrial ATP/ADP ratios can be taken for they are directly regulated by the activity of the ANT. These ratios were measured by nonaqueous fractionation technique in freeze-clamped isolated perfused hearts: In several hearts of virus-infected mice, that all had generated ANT-specific antibodies, ATP/ADP ratios were lowered in the cytosol and elevated in the mitochondria. As the major contribution to the increase in mitochondrial ATP/ADP ratios, the mitochondrial ATP concentration had more than doubled in five hearts of the infected group (Table 2). This observation is highly characteristic for a disturbed function of the ANT [13]. In parallel, cytosolic ATP and creatine phosphate concentrations, as well as mitochondrial ADP concentrations were reduced up to 50% in these hearts. However, in nine hearts of the infected plus antibody-positive group, no changes in energy metabolism were noticed. One explanation for that incomplete functional affection of the ANT might be the polyclonal nature of these antibodies: not all of them being able to influence ANT function.

The reduced cytosolic and elevated mitochondrial ATP/ADP ratios argue against an impairment of oxidative phosphorylation being responsible for the lowered mechanical work, but they may reflect an impaired function of the ANT. We have found an identical pattern of alteration of subcellular myocardial energy metabolism in hearts of guinea pigs that had generated antibodies against the ANT by direct immunization with this protein, but never in failing ischemic hearts [31]. The significance of this specific modulation in myocyte energy metabolism is confirmed by the reduction of cardiac performance, that correlates with the degree of inactivation of the ANT (Figs. 4 and 5Go), and that was similarly observed in ANT-immunized guinea pigs. [14,15] Evidence that reduced energy metabolism is linked to impaired contractile function grows from the observed inefficiency in oxygen utilization in the five hearts with overt metabolic alterations. These hearts show a higher MVO2/LVP and MVO2/RPP ratio than the control collective.

However, these findings do not prove that the ANT-antibodies are responsible for the altered cardiac energy metabolism and the depressed heart function. The virus infection itself or mediator substances of the immune reaction like cytokines may trigger the metabolic and functional changes. How the immune system is modulated by virus infection is not completely resolved. [32] Autoimmunity might be induced by a cross-reactivity of antibodies against viral proteins with myocardial structures, called ‘molecular mimicry’ [33,34]. In our laboratory, we raised cross-reacting antibodies against synthetic peptides from homologous parts of the amino acid sequence of the ANT and the Coxsackie B enterovirus [16].

Whether the antibodies directly bind to the antigen or act indirectly is still a matter of discussion. One hypothesis proposes a change in cellular calcium homeostasis by activating Ca2+-permeable channels [35]. An intracellular calcium overload may lower the transmembrane phosphorylation potential and reduce the nucleotide exchange via the ANT [36–38]. A specific binding of antibodies against the ANT to a myolemnal calcium channel and an antibody-induced enhancement of calcium influx into the cytosol has been demonstrated [39]. Also the virus infection itself or the immune reaction may change the expression of the ANT and therefore a switch of its isoform pattern might change its transport characteristics and alter myocardial energy metabolism [40,41].


   5 Conclusion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
References
 
We showed in a murine model that specific antibodies against the ANT are generated in Coxsackie-B3 virus-induced myocarditis. We further observed that in several of the antibody-positive mice hearts cytosolic ATP/ADP ratios are reduced and mitochondrial ATP/ADP ratios are elevated, thus indicating ANT dysfunction. In addition, heart function is depressed when these signs of an altered ANT activity are present. The degree of the metabolic disorder correlates with the reduction of cardiac performance. The data do not prove that the antibody itself is directly responsible for the noticed metabolic and functional impairments, but they confirm our former observations on guinea pigs immunized with the ANT, whereby their hearts were also functionally affected by carrier-specific antibodies and cardiac energy metabolism was identically changed. Thus, the hypothesis that autoimmunological processes are relevant in myocarditis and in the pathogenesis of dilated cardiomyopathy is strengthened. These findings are an important link between experimental data and clinical observations in myocarditis and in dilated cardiomyopathy.

Time for primary review 27 days.


   Acknowledgements
 
This work was supported by the grants of the Deutsche Forschungsgemeinschaft SFB 189-B8 and SFB 242-E4


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusion
 References
 

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