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European Journal of Heart Failure 2006 8(1):81-89; doi:10.1016/j.ejheart.2005.05.003
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© 2005 European Society of Cardiology

An isoform shift in the cardiac adenine nucleotide translocase expression alters the kinetic properties of the carrier in dilated cardiomyopathy

Andrea Dörnera,*, Stefanie Giessena, Regina Gauba, Helga Große Siestrupa, Peter L. Schwimmbecka, Roland Hetzerb, Wolfgang Pollera and Heinz-Peter Schultheissa

a Charité University Medicine, Campus Benjamin Franklin Department of Cardiology, Berlin, Germany
b Department of Cardiothoracic and Vascular Surgery Deutsches Herzzentrum Berlin, Berlin, Germany

* Corresponding author. Charité-Campus Benjamin Franklin, Department of Cardiology and Pneumology, Hindenburgdamm 30, 12200 Berlin, Germany. Tel.: +49 30 8445 4577; fax: +49 30 8445 3565. E-mail address: andrea.doerner{at}charite.de


   Abstract
Top
 Abstract
1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 References
 
Background: Impaired mitochondrial ADP/ATP transport and altered adenine nucleotide translocase (ANT) isoform expression characterized by enhanced ANT1 and decreased ANT2 expression have been implicated in the pathophysiology of dilated cardiomyopathy (DCM). It is still unknown whether restricted ANT function results from exogenous factors, or mutations in the ANT genes, or whether the imbalance in the isoform composition causes the reduced ADP/ATP transport. We performed DNA mutation screening of ANT genes and analyzed the kinetic properties of ANT protein isolated from DCM hearts and controls in a reconstituted system excluding natural environmental influences.

Results: A G1409T polymorphism in ANT2 leads to an exchange from Arg111 to Leu111 in healthy blood donors (n=60) with allele frequencies of 76% and 24%. This polymorphism was neither associated with DCM (74%, 26%; n=93) nor with altered myocardial ANT isoform expression or restricted ANT function (89%, 11%; n=8). However, there was a remarkable reduction in the maximum transport activity (vmax) of reconstituted ANT from DCM hearts with altered ANT isoform expression (498±113 µmol min–1 g–1 incorporated protein vs. 1112±178 µmol min–1 g–1 incorporated protein, p<0.01). Moreover, the substrate affinity of DCM myocardial ANT to ATP was slightly reduced with an increased Km value of 104.3±2.4 µM vs. 90.4±2.9 µM in controls (p<0.03).

Conclusion: The altered isoform expression in DCM hearts entails changes in the kinetic properties of total ANT protein restricting ANT function and contributing to disturbed energy metabolism in DCM.

Key Words: Adenine nucleotide translocase • Dilated cardiomyopathy • Isoform • Mitochondrial transport

Received November 5, 2004; Revised December 8, 2004; Accepted May 5, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Patients and methods
 3. Results
 4. Discussion
 References
 
The adenine nucleotide translocase (ANT) is a dimeric protein complex of two identical 32 kDa subunits and facilitates the transport of ADP and ATP across the inner mitochondrial membrane [1]. Therefore it is the key link between ATP production in the mitochondrial matrix space and ATP consumption in the cytosol. In addition, ANT plays a significant role in apoptosis as a component of the mitochondrial permeability transition pore [2] and is an important binding partner for pro-, anti-apoptotic [3] and viral proteins [4,5] as well as for transcription factors [6]. ANT is encoded by three different genes (ANT1-3) [7,8] co-expressed in tissue-specific patterns [9]. ANT1 is mainly expressed in tissues with restricted mitotic regeneration like the heart, skeletal muscle and brain. In contrast, ANT2 was found to be growth-regulated and is the prevalent isoform of kidney, liver and spleen, but is also present in high amount in tissue mainly expressing ANT1 [10]. The ANT3 gene with features of a housekeeping gene is transcribed to a lesser degree in each of the tissues. Due to their high homologies of 88-91%, ANT isoform proteins have not yet been separately isolated from human tissue, and therefore little is known about their individual kinetic and functional characteristics.

The identification of ANT-specific antibodies in patients with dilated cardiomyopathy (DCM) provided the first evidence that ANT is involved in the pathophysiology of DCM [11]. The antibodies were organ- and confirmation-specific and inhibited the nucleotide transport of heart mitochondria in vitro. Subsequent studies showed reduced ANT transport capacity in explanted heart tissue of patients with DCM [12]. This impaired ANT function was accompanied by an increase in the amount of ANT protein [12,13]. Consequently, a deficiency in total ANT protein cannot be responsible for the reduced ANT transport capacity in DCM hearts. Such an increase in the amount of myocardial ANT was also found in serotonin receptor knockout mice developing DCM [14]. Further analyses of human DCM hearts revealed a shift in the ANT isoform expression [15]. This ANT isoform shift was characterized by increased ANT1 and decreased ANT2, whereas ANT3 remains unchanged. Altered ANT isoform expression was not only found in explanted heart tissue of DCM patients but also in endomyocardial biopsies taken from patients during diagnostic catheterization. Thus, altered ANT expression is not a feature of explanted hearts representing end stage heart failure but already occurs in the course of disease development. This was also observed in inflammatory heart disease associated with enteroviral infection, which supports the link between viral inflammatory heart disease and DCM [16]. ANT isoform expression was, however, not modified in the heart tissue of patients suffering from other heart diseases such as ischemic, valvular, or hypertrophic cardiomyopathy [15]. This suggests a possible DCM-specific molecular pathomechanism associated with altered ANT function and expression.

It remains to be clarified whether reduced ANT function in DCM hearts is caused by altering ANT isoform expression or whether ANT transport capacity is restricted by exogenous factors or mutations in the ANT genes. To answer these questions, genomic DNA of DCM patients was screened for ANT gene mutations. Moreover, the kinetic properties of human ANT protein extracted from explanted heart tissue with normal or altered ANT isoform expression seen in DCM were analyzed by liposome reconstitution. This system excludes interference from the complex mitochondrial environment in adenine nucleotide transportation.


   2. Patients and methods
Top
 Abstract
 1. Introduction
 2. Patients and methods
3. Results
 4. Discussion
 References
 
2.1. Patients
Blood samples from 93 West European patients with DCM (32 females, 61 males), and 60 healthy blood donors (21 females, 39 males) were used for ANT1 and ANT2 mutation screening. Patients were analyzed according to WHO diagnostic standards. DCM patients had left ventricular ejection fractions (EF) of less than 45 and left ventricular end-diastolic diameters (LVEDD) of more than 58 mm. Patients with the following conditions were excluded from the DCM group: ischemic, valvular or hypertensive disease, congenital malformations, fibroelastosis, hemochromatosis, neuromuscular disease or acute myocarditis confirmed by endomyocardial biopsies.

Left ventricular tissue samples were obtained during heart transplantation from excised hearts of heart recipients for ANT isolation and reconstitution. The myocardial ANT isoform percentages of enrolled patients were pretested according to the method described elsewhere [9]. Normal myocardial ANT isoform expression (nANT) is characterized by a percentage of 68.1±4.5% for ANT1, 24.9±5.0% for ANT2 and 7.0±2.7% for ANT3 [15]. The resulting ANT1/(ANT2+ANT3)mRNA ratio of 2.2±0.5 describes this relationship as a single value for people with no heart failure and patients with ischemic or valvular cardiomyopathy [15]. Specimens from 9 DCM patients pretested as having an altered ANT (aANT) isoform expression with an ANT1/(ANT2+ANT3)mRNA ratio of ≥3.7 (mean(control)+3xSD) were included in the present study. We used heart tissue from 8 patients with a normal ANT isoform expression (nANT), a isoform ratio of <3.7 and end-stage heart failure due to ischemic cardiomyopathy as controls. Biographical and hemodynamic data of both groups are summarized in Table 1.


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  		Table 1 Biographical and hemodynamic data of patients with normal myocardial ANT isoform expression (nANT) and ischemic cardiomyopathy and from DCM patients with an altered ANT isoform expression (aANT)

 
The investigation conforms with the principles outlined in the Declaration of Helsinki and all patients gave their written informed consent. Explanted heart tissue was immediately snap-frozen in liquid nitrogen after removal and stored at –80 °C.

2.2. SSCP mutation screening
DNA was isolated from blood and heart tissue using the QIAamp Blood Kit (Qiagen, Hilden, Germany) or the E.Z.N.A. Tissue DNA Mini Kit (peqLab, Erlangen, Germany). The coding ANT1- [7] and ANT2-specific gene sequence [8] was amplified from position –555 to +4100 and from –1927 to +3038 by 12 PCR products not larger than 450 bp using AmpliTaq DNA polymerase (Roche, Branchburg, USA) according to the manufacturer's instructions. Mutation screening was performed by the single-strand conformation polymorphism (SSCP) technique as described by Orita et al. [17,18]. A polymorphism in the ANT2 gene was found between position +1165bp and +1536bp amplified using primers 5'-CCCTGTTGGCTTCCTTCCTG and 5'-TTCCCTTTCAGCTCCAGCTT-3'. Nucleotide exchange alters the HaeII restriction site used for the restriction fragment length polymorphism method to confirm genotype frequencies.

2.3. Antibody production
250 µg of purified myocardial bovine ANT (kindly donated by Prof. Beyer, Physiological Biochemistry, Munich) or 200 µg of KLH-coupled ANT1-specific peptide (KGAAQREFHG) were used for antibody production in New Zealand white rabbits according to a standard protocol.

2.4. Antibody specificity
Antibody specificity was tested by ELISA as previously described elsewhere [19]. ELISA plates were coated overnight with 10 µg/ml of ANT1-, ANT2- or ANT3-specific peptides (ANT1: KGAAQREFHG; ANT2: KAGAEREFRG; ANT3: KSGTEREFRG) each located in the AA146-AA155 region of the ANT isoform proteins. Antisera against total ANT protein or ANT1-specific peptide were each diluted 1:10 and subsequently used in fourfold dilutions in the ELISA (Fig. 1A). Total mitochondrial membrane protein and purified myocardial bovine ANT protein were fractionated on 12% SDS-polyacrylamide gels, and Western blot was done with antibodies against ANT1 and total ANT protein according to a standard protocol (Fig. 1B).


Figure 1
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  		Fig. 1 (A) Specificity of the antibodies directed against (a) total myocardial ANT protein and (b) against human ANT1-specific protein was tested by the ELISA technique. ANT1- (bullet), ANT2- (*) and ANT3-specific peptides (+) with amino acid sequences from the same region of each ANT isoform (AS146-155) were used as antigens. Antibodies against myocardial ANT interacted with all ANT isoform-specific peptides. In contrast, ANT1-specific antibodies bound exclusively to the ANT1-specific peptide. (B) Protein molecular weight marker (MW), purified myocardial bovine ANT protein (ANT) and human total membrane protein (MP) were subjected to SDS gel electrophoresis and (a) stained with Coomassie blue. Proteins were blotted onto nitrocellulose membranes and western blot analyses were performed using antibodies against the total myocardial ANT protein (b) and against the human ANT1 protein (c). Both antibodies specifically recognized the myocardial bovine as well as the human ANT protein and showed no cross-reactivity with other proteins.

 
2.5. Immunodot blot
Mitochondria were isolated from the heart tissue according to Smith [20]. Mitochondrial proteins were extracted in 0.1 M Na2SO4, 10 mM Mops, pH 7.2, 1% Triton X-100, or reconstituted ANT protein from liposomes was precipitated with trichloroacetic acid and dissolved in extraction buffer. After centrifugation, dilutions of mitochondrial or reconstituted proteins were spotted onto two nitrocellulose membranes. A dilution of purified myocardial bovine ANT protein or ANT1 peptide was additionally applied to one of the membranes in a defined concentration and was used as the standard. The membranes were blocked and incubated with antiserum against total ANT protein or ANT1 protein, washed and incubated for 2 h in PBS, 1% Tween 20 containing 10 ng/ml of 35S-protein A. Bound 35S-labeled protein A of each dot was measured in a beta counter. The standard curves were determined by linear regression and each sample was compared to the standard.

2.6. Reconstitution of the ANT protein and determination of its kinetic properties
The ANT protein was extracted from mitochondria by hydroxyapatite chromatography in a batch procedure using the detergent C12E8 as described by Riccio et al. [21].

The ANT protein was reconstituted according to the method described by Krämer et al. [22,23]. Liposomes were formed by sonification of a mixture containing 12 mg of phospholipids/ml reaction volume, 0.01 mg of ANT protein/mg phospholipids, 1.5 mg of C12E8/mg phospholipids in 50 mM Na2SO4, 20 mM Tricine/NaOH (pH 7.5), and 20 mM ATP as a substrate for 20 min in an argon atmosphere at 4 °C. The mixture was passed through Amberlite columns 14 times to continually remove the detergent. Subsequently, external ATP was cleaned off of proteoliposomes by passing the vesicles through a gel chromatography column. The amount of ANT protein incorporated into the liposomes was determined according to the method described by Wessel and Flügge [24].

To prevent the formation of a pH gradient or membrane potential, the proteoliposomes were mixed with valinomycin (500 ng/mg of phospholipids) and nigericin (200 ng/mg of phospholipids). Transport was started by adding 10 µl of ATP at concentrations of 0.63, 3.13, and 6.27 14C-ATP and 10, 20, 50 and 100 µM nonradioactive ATP each mixed with 1.6 µM 14C-ATP (14C-ATP; specific activity: 53 mCi/mmol) to the proteoliposomes. The exchange was stopped using a mixture of 40 mM pyridoxalphosphate and 1 mM atractyloside after 0.5, 1, 2, 4 and 8 min. In the control samples, the inhibitors were added with the substrate at the beginning. Unincorporated radioactivity was removed from the vesicles by passing them through anion exchange columns with Dowex 2-X10 and Dowex 1-X8 at a ratio of 1:8. The columns were pre-equilibrated with 1% phospholipids and 1% bovine serum albumin, and vesicles were loaded onto the columns and rinsed by subsequent washing steps of 100, 300 and 500 µl of 300 mM sucrose. Endogenous radioactivity was measured in a beta counter. The transport activity was calculated using a computer fitting program (Enzfitter-Biosolf) adjusting the time course of isotope equilibration to the incorporated radioactive-labeled substrate. The Eadie-Hofstee plot was used to determine the Km value (µM) and vmax expressed as µmol ATP/min/g of incorporated ANT protein.

2.7. Statistics
The Student's t-test or the Mann-Whitney U test for unpaired samples was used for statistical analysis of the data. Values are shown as the mean±SEM of n independent experiments except when otherwise stated. The Chi2-test was used to analyze the statistical significance of the association between genotype and heart diseases. A p-value of <0.05 showed statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
4. Discussion
 References
 
3.1. Polymorphism of ANT2 gene
The coding regions of human ANT1 and ANT2 genes were screened for mutations that might affect the kinetic properties of the ANT isoform proteins. No alterations were found in the coding nucleotide sequence of ANT1. However, an exchange from guanine to thymine was detected in exon II at position +1409bp in the X-chromosomal ANT2 gene that causes an amino acid exchange from basic Arg111 to an aliphatic Leu111. The genotype distribution in the different groups complies with the Hardy-Weinberg equilibrium. Genotype frequency analyses showed Arg111 to be the more frequent allele in the group of healthy blood donors and DCM patients with equal frequency of the alleles in females and males (Table 2A,B). The genotype distribution did not differ between blood donors and DCM patients.


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  		Table 2
 
3.2. ANT isoform expression
For ANT reconstitution, we used heart tissue from eight patients with a normal ANT isoform expression characterized by an average ANT1/ANT2+ANT3 mRNA ratio of 2.1±0.3 (Table 3). In addition, hearts of nine DCM patients with an altered ANT isoform expression (aANT) and an elevated isoform ratio of 5.7±2.5 were analyzed. The shift in the ANT isoform ratio was based on an elevated ANT1 and a reduced ANT2 percentage.


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  		Table 3 ANT expression data of patients with normal (nANT) and altered (aANT) cardiac ANT isoform expression whose heart tissue was used for ANT reconstitution

 
Total ANT protein correlated strongly to alterations in the ANT1/(ANT2+ANT3) mRNA ratio with a correlation factor of 0.81 (p<0.001) (Fig. 2). The amount of mitochondrial ANT1-specific protein was almost doubled in DCM hearts, whereas the increase in total ANT protein was only 43.6%, indicating that ANT2 protein is simultaneously decreased as seen for the percentage of ANT2 mRNA (Table 3). Thus, changes in the ANT isoform mRNA ratio are reflected at the protein level.


Figure 2
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  		Fig. 2 The ANT isoform mRNA ratio was correlated to the amount of total myocardial ANT protein of heart samples from patients with a normal myocardial ANT isoform expression ({circ}) and those with an altered ANT isoform expression (bullet).

 
ANT2 allele frequencies did not differ in the two groups (Table 3), which means that altered ANT isoform distribution is not related to a single ANT2 allele.

3.3. Reconstitution of the ANT protein
The kinetic characteristics of total ANT protein with a normal and an altered ANT isoform composition were each determined independently of the natural mitochondrial environment by reconstituting purified ANT in phospholipid vesicles. Isolated ANT protein had an 86% purity (Fig. 3A) determined by densitometric analysis. The 34 kDa protein slightly contaminating the preparation is known as porin [22]. Empty liposomes did not facilitate any ATP/ATP homoexchange across the artificial membrane. ANT incorporation leads to a substrate exchange that was almost completely abolished by pre-incubation with specific ANT inhibitors, which demonstrates the reliability of the system (Fig. 3B). The ANT1 percentage of liposomes determined for three specimens of each group correlated strongly with that of the respective myocardial mitochondria, proving that extracted ANT isoforms were equally extracted from mitochondria and incorporated into liposomes (Fig. 3C).


Figure 3
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  		Fig. 3 (A) Reconstituted ANT protein with a normal (nANT) and an altered (aANT) ANT isoform expression was subjected to SDS gel electrophoresis and was stained with Coomassie blue. Preparations are slightly contaminated with porin. (B) The time course of isotope equilibration using liposomes with incorporated ANT ({square}) and those without ANT protein ({blacktriangleup}). The velocity of C14-ATP uptake was almost completely blocked by ANT-specific inhibitors ({blacksquare}). (C) ANT protein was isolated and reconstituted from the heart tissue of controls ({circ}) and DCM patients with an altered ANT isoform expression (bullet). The ANT1 protein percentage of liposomes significantly correlated to that of the respective mitochondria.

 
There was a significant decrease of 55% in maximum specific transport activity vmax of ANT with an altered ANT isoform ratio compared to controls (Table 4). Moreover, the average Km value of aANT protein was slightly higher than that for controls. The amount of mitochondrial ANT1 correlated positively with increasing Km and negatively with decreasing vmax values underlining the link between ANT isoform ratio and kinetic properties of the complete myocardial ANT protein (Fig. 4). The polymorphism found in the ANT2 gene is not relevant for the changed kinetic properties of ANT, since only one patient of each group had the Leu111 allele (Table 3).


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  		Table 4 Kinetic characteristics of total myocardial ANT protein with normal and altered ANT isoform expression

 


Figure 4
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  		Fig. 4 (A) ANT1 protein amount was correlated with Km and (B) vmax values of the reconstituted ANT protein from patients with a normal ({circ}) and an altered ANT isoform expression (bullet).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
References
 
Nuclear gene mutations expressed in heart tissue have been identified as the molecular basis of the disease in small, monogenic subgroups of DCM [25]. Several studies have reported mtDNA defects in DCM patients, showing that genetic modifications play a role in the origin of the disease [26]. Furthermore, ANT1 mutations or restricted ANT1 expression have been associated with diseases like autosomal dominant progressive external ophthalmoplegia [27] or Sengers syndrome [28]. We therefore analyzed the coding sequences of the human ANT1 and ANT2 genes. A G1409T mutation in the ANT2 gene causes an exchange of basic Arg to lipophilic Leu at amino acid position 111 in the third transmembrane domain of the ANT2 protein. Since this polymorphism is not rare in the general population considering the X-chromosome linkage of ANT2 and a Leu111 frequency of 24%, we conclude that Leu111 does not negatively affect ANT2 function. There was no association between Leu111 and reduced ANT transport activity, an altered ANT isoform expression or DCM, which thus excluded point mutations in the ANT genes as a possible explanation for restricted ANT function in DCM hearts. Interestingly, no polymorphism was found in the third transmembrane region of the ANT1 gene. Mutations in this region have been associated with the mitochondrial disorder adPEO [27,29] and with swelling and disintegration of mitochondria in yeast [30]. Thus, ANT1 protein variations may be very critical for its function and do not appear in the general population.

Reconstituting transport processes across artificial membranes elucidates the kinetic characteristics of the carrier under strictly defined conditions without interference from the mitochondrial environment like oxidative phosphorylation, Ca+2 content, or protein-protein interaction. The isolated ANT protein used for the reconstitution was of equal quality, and the Km and vmax value of human ANT protein from the controls corresponded to that of myocardial bovine ANT of 1000 µmol of ATP min–1 g–1 incorporated protein [31]. Mitochondrial oxidative damage influencing ANT activity has been described in both ischemic and dilated cardiomyopathy [32,33] and is unlikely to be responsible for the restricted ANT function found in DCM but not in ischemic cardiomyopathy.

The Km value of whole ANT protein isolated from hearts with an altered ANT isoform ratio was slightly increased compared to controls. An elevation in the Km value indicates a lower affinity of the carrier to its substrate and a reduced stability of the substrate-carrier complex. As shown in Fig. 4, a greater amount of ANT1 protein in the liposomes increases Km . However, it is still unclear if physiological significance can be attributed to the increased Km value in ANT isolated from DCM hearts, since the concentration of ATP in mammalian mitochondria is about 80 times higher than the Km of the ANT protein [34].

The increase in ANT1 and decrease in ANT2 causes a remarkable reduction in the maximum transport activity of the reconstituted ANT protein. It has long been assumed that ANT1 is the isoform most suited for catalyzing rapid exchange of ATP and ADP across the mitochondrial membrane, since it is predominantly expressed in high energy-consuming tissues like heart, muscle and brain. In contrast to this assumption, our data showed a significant correlation between an increasing ANT1 respectively decreasing ANT2 amount and decreasing vmax values, which led to the conclusion that the transport activity of ANT1 is lower than that of ANT2. This is underlined by studies of De Marcos Lousa et al. [35] who demonstrated significant differences in the kinetic properties of human ANT isoforms expressed and analyzed in the complex, unnatural environment of yeast mitochondria. The importance of ANT2 as the actual high-capacity isoform for energy transfer is demonstrated by the fact that the amount of ANT2 is substantially higher in tissues with extreme energy consumption such as heart, brain, and muscle than in other organs even there is a high ANT1 expression [10]. The high efficiency of ANT2 in energy transfer was also clearly shown in ANT1 knockout mice [36]. ANT2 alone was found to maintain 86% of the ADP-stimulated mitochondrial respiratory rate in heart tissue from ANT1 knockout mice with completely eliminated ANT1 and stable ANT2 expression.

Differences in the kinetic properties of the ANT isoforms may be justified in different localization within the mitochondrium, different interaction with kinases and/or distinct additional functions besides ADP/ATP transportation. The peripheral inner mitochondrial membrane contains ANT1 and ANT2, whereas the cristal membrane only includes ANT2 [37]. ANT1 but not ANT2 was found to bind to hexokinase, whereas both isoforms were detected in complexes with mitochondrial creatine kinase [37]. With other proteins, ANT forms pores known as mitochondrial permeability transition pores (MPTP), whose opening leads to mitochondrial swelling and release of several apoptosis-inducing factors like cytochrome c and apoptosis-inducing factor AIF. The affinity of cyclophilin, an apoptosis-regulating and MPTP-interacting protein, appeared to be higher to ANT1 than to ANT2 [37]. Moreover, the overexpression of ANT1 and ANT3 has been shown to induce apoptosis in HeLa cells that have a naturally predominant ANT2 expression, whereas ANT2 did not show this effect [38,39]. These findings lead to the current discussion that ANT1 might be more relevant for inducing apoptosis than ANT2. In addition, ANT1 is the predominant binding partner for the BART-ARL2-GTP complex, whereas ANT2 did not bind to this protein complex involved in cellular communication [40]. This strengthens the hypothesis that ANT1 is not only a transporter for energy-rich phosphates but also an important mitochondrial regulator of intracellular processes.

The pathophysiological impact of deficient ATP/ADP transport has been demonstrated in ANT1 knockout mice [36]. The heart tissue of these animals revealed cardiac hypertrophy with mitochondrial proliferation and degeneration. The mice manifested serious exercise intolerance with severe myopathy and cardiomyopathy. In addition, mitochondria isolated from ANT1-deficient mice produced a markedly increased amount of reactive oxygen species that damage mtDNA and cause cellular dysfunction. Several mitochondrial genome mutations were also found in DCM patients. Pathological mtDNA mutations are associated with ultrastructurally abnormal mitochondria and disturbed energy metabolism like that observed in DCM hearts.

In summary, we excluded ANT point mutations as a reason for restricted ANT function in DCM hearts. We have shown that the altered ANT isoform expression in the myocardium of DCM patients entails changes in the kinetic properties of the entire ANT protein. Thus, doubling of ANT1 expression cannot compensate the reduced amount of ANT2 in DCM that is most relevant for ADP/ATP translocation across the inner mitochondrial membrane. Since cardiac ANT dysfunction causes disturbed energy metabolism and impaired heart function, the isoform shift appears to contribute to cardiac dysfunction in DCM.


   References
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 References
 

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