Alterations in myocardial tissue factor expression and cellular localization in dilated cardiomyopathy
Björn Szotowski, MPharm*, ,
Petra Goldin-Lang, PhD*,
Silvio Antoniak, MS*,
Vladimir Y. Bogdanov, PhD ,
Delano Pathirana*,
Matthias Pauschinger, MD*,
Andrea Dörner, PhD*,
Uwe Kuehl, PhD*,
Sarah Coupland, MD ,
Yale Nemerson, MD,
Michael Hummel, MD,
Wolfgang Poller, MD*,
Roland Hetzer, MD, PhD||,
Heinz-Peter Schultheiss, MD* and
Ursula Rauch, MD*,*
* Department of Cardiology and Pneumology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
Institute of Pharmacy, Free University of Berlin, Berlin, Germany
Department of Medicine, Mount Sinai School of Medicine, New York, New York
Institute of Pathology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
|| Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrum Berlin, Berlin, Germany
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Figure 1 Gel electrophoresis (1% agarose gel) of reverse transcription-polymerase chain reaction products (human tissue factor [hTF] and alternatively spliced human tissue factor [asHTF]) using ribonucleic acid from human heart biopsies. (M) 250 bp deoxyribonucleic acid ladder; (C) no template, control; (1) explanted heart tissue (ventricular septum); (2) human myocardial biopsy from patient with normal cardiac function; (3) human myocardial biopsy from patient with severely impaired cardiac function.
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Figure 2 Western blot for tissue factor isoforms from explanted dilated cardiomyopathy hearts. Extracts from explanted dilated cardiomyopathy hearts were immunoprecipitated with anti-human tissue factor (TF) antibodies directed against the TF extracellular domain. Western blots showed both, full-length TF and alternatively spliced human tissue factor (asHTF) (lane 1), detected by a polyclonal anti-TF antibody (pAb-sTF) directed against the extracellular domain of the TF protein. Rehybridization of the membrane with a polyclonal antibody (asHTF), directed against the last 29 amino acids of the unique asHTF end yielded a single protein band at approximately 30 kDa, whereas full length TF was not detected (lane 2).
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Figure 3 Immunohistochemistry for tissue factor (TF), alternatively spliced human tissue factor (asHTF), and desmin. Polyclonal rabbit antibodies directed against the extracellular domain of TF and the unique C-terminal end of asHTF were used for staining of the myocardial biopsies obtained from a patient with normal ejection fraction (EF) (64%) and a patient with dilated cardiomyopathy (DCM) (21%). (A and B) Cardiomyocytes and endothelial cells within the normal myocardium stained positive for asHTF. Original magnification 400x. (C) Immunohistochemistry for desmin showed positive staining of Z-bands of the cardiomyocytes in a patient with normal EF. The sarcolemma and Z-bands of the same patient were also positive for TF (arrows, D). Original magnification 630x. (E) The Z-bands stained also positive for desmin in the myocardium of a patient with DCM. In contrary to desmin, TF staining of the Z-bands was faint or completely absent in the myocardial biopsy of the same DCM patient (arrows, F and G). Myocardial TF was located within the cytosol around the nucleus in DCM (arrow heads, F and G). Original magnification 630x. (H) Control for TF staining. Original magnification 630x. (I) TF staining of the Z-bands of a patient with normal EF without hematoxylin. Original magnification 630x. (J) Immunohistochemistry for TF showed a diffuse staining with absence of TF-positive Z-bands. TF was located around vacuoles (arrowheads). Original magnification 630x.
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Figure 4 Quantification of tissue factor (TF) expression in ventricular septum of explanted dilated cardiomyopathy (DCM) hearts (n = 12) and of biopsied patients with DCM (n = 27) and with normal to mildly reduced ejection fraction (EF) (n = 19). Data are given as mean ± SD. (A) Full-length TF/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger ribonucleic acid (mRNA) ratio; (B) Alternatively spliced TF/GAPDH mRNA ratio. asHTF = alternatively spliced human tissue factor.
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Figure 5 Graphs demonstrate correlations between tissue factor (TF) isoform expression and ejection fraction (EF). (A) Regression line between the messenger ribonucleic acid (mRNA) TF/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio and EF (r = 0.504, p < 0.0001). (B) Regression line between the mRNA alternatively spliced human tissue factor (asHTF)/GAPDH ratio and EF (r = 0.382, p = 0.003).
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Figure 6 (A) Quantification of tissue factor (TF) protein (ng TF protein/mg total protein) from biopsied patients with dilated cardiomyopathy (n = 15) and normal to mildly reduced ejection fraction (EF) (n = 18). Data are given as mean ± SD. (B) Correlation between TF protein and EF (r = 0.423, p = 0.014).
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