Identification of mitochondrial antigens recognized by antibodies in sera of patients with idiopathic dilated cardiomyopathy by two-dimensional gel electrophoresis and protein sequencing.
Antimitochondrial antibodies in sera of patients with idiopathic dilated cardiomyopathy (IDC) have been described previously, but the corresponding antigens have not been analyzed systematically. We therefore used both 1-dimensional and high-resolution 2-dimensional gel electrophoresis followed by immunoblotting and N-terminal amino acid sequencing to identify the relevant mitochondrial antigens, which are recognized by serum antibodies. Sera were obtained from patients with IDC (n = 75) and healthy controls (n = 182). For detection of antimitochondrial antibodies the mitochondrial antigen fraction, consisting of submitochondrial particles isolated from a bovine heart, was separated on SDS-PAGE and all sera were examined by immunoblot analysis. For further characterization of the mitochondrial epitopes the antigen fraction was separated in the first dimension according to isoelectric points using isoelectric focusing followed by gel electrophoresis. Proteins recognized by serum antibodies in 2-dimensional immunoblots were analyzed by N-terminal amino acid sequencing. In 1-dimensional immunoblot analysis, 51% of patients with IDC and 34% of controls contained serum antibodies reacting with mitochondrial protein bands with molecular weights of about 30, 43, 60, and preferentially 50 to 55 and 70 to 75 kD (p <0.01). We identified a 75-kD subunit of nicotinamide-adenine dinucleotide dehydrogenase (17% in IDC patients vs 5% in controls, p <0.05) and 2 core proteins of ubiquinol-cytochrome-c reductase (core P1: 39% in IDC patients vs 15% in controls, p <0.05; core P2: 20% in IDC patients vs 10% in controls, p <0.1), both enzymes of the respiratory chain, as are most relevant mitochondrial antigens. Furthermore, serum antibodies of patients with IDC were directed against lipoamide-dehydrogenase (15% vs 10% in controls) and a subunit of pyruvate-dehydrogenase (9% vs 3% in controls). Because these antigens play an important role in energy metabolism, the respective antibodies can be more than merely diagnostic markers of cell damage. To attribute them also to pathogenetic relevance appears to be a most attractive but still speculative hypothesis.
Pohlner
K
K
Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany.
Portig
I
I
Pankuweit
S
S
Lottspeich
F
F
Maisch
B
B
D000328:Adult; D000595:Amino Acid Sequence; D001324:Autoantigens / Q000097:blood*; D002311:Cardiomyopathy, Dilated / Q000276:immunology*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000276:immunology*; D008969:Molecular Sequence Data
D001324:Autoantigens