lterations in Myocardial Tissue Factor Expressionnd Cellular Localization in Dilated Cardiomyopathyjörn Szotowski, MPHARM,*† Petra Goldin-Lang, PHD,* Silvio Antoniak, MS,*ladimir Y. Bogdanov, PHD,‡ Delano Pathirana,* Matthias Pauschinger, MD,* Andrea Dörner, PHD,*we Kuehl, PHD,* Sarah Coupland, MD,§ Yale Nemerson, MD,‡ Michael Hummel, MD,§olfgang Poller, MD,* Roland Hetzer, MD, PHD,� Heinz-Peter Schultheiss, MD,* Ursula Rauch, MD*
erlin, Germany; and New York, New York
OBJECTIVES We investigated the myocardial localization and expression of tissue factor (TF) andalternatively spliced human tissue factor (asHTF) in patients with dilated cardiomyopathy(DCM).
BACKGROUND Tissue factor is expressed in cardiac muscle and may play a role in maintaining myocardialstructure.
METHODS Myocardial biopsies were obtained from patients with a normal or mildly impaired ejectionfraction (EF) (�50%) and moderate to severely reduced EF (�50%). Explanted DCM heartswere also examined. Myocardial TF expression level was assessed by real-time polymerasechain reaction, TF protein by enzyme-linked immunosorbent assay, and localization byimmunohistochemistry.
RESULTS We report the identification of asHTF in the human myocardium: it was located incardiomyocytes and endothelial cells. Quantification of myocardial TF messenger ribonucleicacid in DCM revealed a decrease in the TF/glyceraldehyde-3-phosphate dehydrogenase(GAPDH) ratio (1.76 � 10�1 � 6.08 � 10�2 for EF �50% [n � 19] vs. 1.06 � 10�1 �5.26 � 10�2 for EF �50% [n � 27]; p � 0.001) and asHTF/GAPDH ratio (13.91 � 10�5
� 11.20 � 10�5 for EF �50% vs. 7.17 � 10�5 � 3.82 � 10�5 for EF �50%; p � 0.014).Tissue factor isoform expression level was also decreased in explanted DCM hearts (p � 0.01;n � 12). Total TF protein was reduced by 26% in DCM (p � 0.05). The TF/GAPDH ratiocorrelated positively with the EF (r � 0.504, p � 0.0001). Immunohistochemistry showedTF localized to the sarcolemma and Z-bands of the cardiomyocytes in patients with normalEF, whereas TF was found in the cardiomyocytic cytosol around the nucleus in DCM.
CONCLUSIONS Tissue factor was down-regulated in the myocardium of DCM patients. The reduction in TFexpression and change in localization may influence cell-to-cell contact stability andcontractility, thereby contributing to cardiac dysfunction in DCM. (J Am Coll Cardiol
ublished by Elsevier Inc. doi:10.1016/j.jacc.2004.12.061
issue factor (TF), the primary initiator of the extrinsicoagulation cascade, is a 263 amino acid transmembranerotein, which is the cellular receptor for factor VII (1–3).eyond its role in hemostasis, TF is abundantly expressedy cardiomyocytes in the human heart but not in skeletaluscle (4,5). In the adult heart, TF is present in the
ransverse part of the intercalated disk and co-localizes withhe cytoskeletal proteins desmin and vinculin (4). Theytoplasmic domain of TF interacts with actin-bindingrotein 280 (filamin), thereby influencing adhesion andigration of cells (6). The myocardial TF expression was
ound to correlate with the number of contact sites ofardiomyocytes, pointing to an important role of TF foraintaining the structural integrity and contractility of theyocardial muscle (4,5). In patients with hypertension and
From the *Department of Cardiology and Pneumology, Charité-Universitäts-edizin Berlin, Campus Benjamin Franklin, Berlin, Germany; †Institute of Phar-acy, Free University of Berlin, Berlin, Germany; ‡Department of Medicine, Mount
inai School of Medicine, New York, New York; §Institute of Pathology, Charité-niversitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany; and
Department of Cardiothoracic and Vascular Surgery, Deutsches Herzzentrumerlin, Berlin, Germany. This work was supported by grants from the Germanesearch Foundation (DFG-SFB/TR 19 and DFG-Graduiertenkolleg 865).
tManuscript received September 13, 2004; revised manuscript received November
9, 2004, accepted December 6, 2004.
entricular hypertrophy, the TF level was found to beeduced in structurally altered ventricular myocardium (4).n addition, a complete TF knockout in the murine 129/Svenetic background leads to TF null embryos that do noturvive beyond mid-gestation (7). Defects in cytoskeletalnd junctional proteins in dilated cardiomyopathy (DCM)re known to contribute to cardiac dilation and dysfunction8). Whether alterations in the myocardial TF expressionlso occur in the cardiac muscle of patients with DCM hasot been examined to date. However, mice expressing low
evels of human TF showed a myocardial defect withyocardial fibrosis, hemosiderin deposition, and ventricular
ysfunction (9). In addition, alternative splicing of the TFre-messenger ribonucleic acid (mRNA) leads to the gen-ration of a soluble TF isoform (asHTF), which has recentlyeen detected in the circulating blood (10). Leukocytic cellsave been suggested to be the source of TF and asHTF inlood (10). Stimulated endothelial cells also express TFRNA isoforms (11) and therefore may contribute to
sHTF in blood as well. Growth factors and cytokines arenown to induce the TF expression in a variety of cardio-ascular cells (11–13). Moreover, cytokines may also con-
ribute to the development of DCM (14). Whether asHTF
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1082 Szotowski et al. JACC Vol. 45, No. 7, 2005Altered TF Expression in Cardiomyopathy April 5, 2005:1081–9
s constitutively expressed in the human myocardium isnknown. Therefore, we examined the expression andocalization of TF and its soluble isoform in the cardiacissue of patients with a normal ejection fraction (EF) and ofatients with DCM.
ATERIALS AND METHODS
tudy design. Myocardial biopsies of 79 patients with theresumptive clinical diagnosis of cardiomyopathy and ven-ricular tissue from 12 explanted DCM hearts were exam-ned. Tissue specimens were taken from the right ventriculareptum and immediately frozen in liquid nitrogen in ordero preserve RNA and protein content. Coronary angiogra-hy was performed in all patients to rule out coronary arteryisease. The presence of valvular heart disease, amyloidosis,r other accumulation disease was also excluded. Accordingo the 1995 World Health Organization/International So-iety and Federation of Cardiology report on the definitionnd classification of cardiomyopathies, 79 biopsied patientsere divided into two groups dependent on the EF mea-
ured during left ventricular catheterization: 37 patients hadormal to mildly reduced EF (�50%) and 42 patientsoderate to severely reduced EF (�50%). The patientsith normal EF had loco-regional wall motion disturbances
n at least two different wall segments on echocardiography
F 5=-TGATGTGGATAAAGGAGAAAACTACTsHTF 5=-GGGATGTTTTTGGCAAGGACTTAAPDH 5=-CCACCCATGGCAAATTCC
C. Probe Sequences
F 5=-FAM-TTCsHTF 5=-FAM-AATAPDH 5=-FAM-TGG
arentheses: amplicon size of asHTF.asHTF � alternatively spliced human tissue factor; GAPDH � glyceraldehyde-3-phos
nd/or had clinical symptoms such as unexplained chestain or heart rhythm disturbances. Ventricular septum ofxplanted hearts from patients with DCM and severelympaired myocardial function was also examined in aeparate group (n � 12). To keep the amount of myocardialiopsies as low as possible and to avoid unnecessary risks forhe patients, either TF mRNA or protein levels wereetermined from a myocardial biopsy. To assess the impactf cardiomyopathy on TF expression, mRNA levels wereuantified by real-time polymerase chain reaction (PCR) in6 of 79 biopsied patients (n � 27 with an EF �50% and� 19 with an EF �50%) and in the explanted hearts (n12). The content of TF protein in myocardial biopsies
as measured by TF enzyme-linked immunosorbent assayELISA) in 33 of the 79 biopsied patients (n � 15 with anF �50% and n � 18 with an EF �50%). Immunohisto-
hemistry was performed to visualize TF protein andocalization within the myocardial tissues. All proceduresere approved by our institutional review board and per-
ormed in accordance with ethical standards and the Hel-inki Declaration of 1975. All patients gave informedonsent for the study.everse transcription-PCR (RT-PCR). Total RNA ofuman heart biopsies was isolated by the TRIzol methodccording to manufacturer’s instruction (Invitrogen,arlsruhe, Germany), reverse transcribed, and analyzed byCR using primers listed in Table 1A. Products wereeparated by electrophoresis on 1% agarose, visualized withthidium bromide under ultraviolet light, and bands werexcised. To ensure that the products obtained by transcrip-ion represented TF isoform sequences, full-length TF asell as asHTF (Gene Bank accession number AF 487337)
pecific sequences were verified by direct sequencing of theCR products. Conventional PCR was performed under
he following conditions: 94°C, 2 min; 94°C, 15 s; 64°C,0 s; 68°C, 1 min for 30 cycles.
1083JACC Vol. 45, No. 7, 2005 Szotowski et al.April 5, 2005:1081–9 Altered TF Expression in Cardiomyopathy
F isoform specific real-time PCR (TaqMan). TotalNA was isolated as described previously. First, 0.5 �g totalNA of each sample was transcribed into complementaryeoxyribonucleic acid (DNA) by using avian mycoblastosisirus reverse transcriptase (AMV RT) and random hexamerrimers according to the supplier’s instructions (Rochepplied Sciences, Mannheim, Germany). Then, 3 �l of
ach complementary DNA preparation was diluted to anal PCR volume of 25 �l containing 12.5 �l TaqManniversal Master Mix (Applied Biosystems, Foster City,alifornia), and 0.25 �l (200 nM) primers (TIB Molbiol,erlin, Germany) and 0.3 �l (100 nM) probes as listed inables 1B and 1C. Each sample was tested for the followingRNAs: TF, asHTF, and GAPDH. In order to differen-
iate between TF and asHTF, primers and probe foretection of TF have been positioned in exon 5, which isissing in asHTF, whereas asHTF primers have been
ositioned in exon 4 and 6, respectively, with the probepanning exon 4/6 boundary, which is not present in TF.eal-time PCR was performed using ABI Prism 7000equence Detection System (Applied Biosystems) under theollowing conditions: 50°C, 2 min; 95°C, 10 min; 40 cycles5°C, 15 s, 60°C, 1 min. Standards covering the completeoding sequence for each target were generated by RT-CR. Serial dilutions of each standard were made.mmunohistochemistry. For TF and asHTF staining,pecimens were immediately snap-frozen and stored at80°C. Cryostat sections were washed with phosphate-
uffered saline, incubated in 4% H2O2, and immunostainedither with polyclonal goat anti-human antibody against TFAmerican Diagnostica Inc., Stamford, Connecticut), di-uted 1:200 in phosphate-buffered saline/fetal calf serum, orith polyclonal rabbit anti-human antibody against asHTF,
espectively. Polyclonal asHTF antibodies were raisedgainst the last 29 amino acids of the unique C-terminalsHTF domain, conjugated to keyhole limpet hemocyaninPineda Antikörper-Service, Berlin, Germany). A secondolyclonal antibody specific for asHTF as previously de-cribed (10) was also used for staining. Myocardial stainingatterns for asHTF obtained by these two antibodies weredentical. After incubation with primary antibody, speci-
ens were washed, incubated with an appropriate biotinyl-ted secondary antibody (DakoCytomation, Hamburg,ermany), and counterstained with hematoxylin (Merck,armstadt, Germany). For detection, the Vectastain ABC
it was used according to manufacturer’s instructions (Vec-or Laboratories, Burlingame, California).
Alternatively, immunohistochemistry was performed onaraffin-embedded tissue blocks from biopsies. Severallides made from every block were deparaffinated overnighty incubation with Roti-Histol (Roth, Karlsruhe, Ger-any). After rehydration they were incubated in a trypsin
olution (1 mg/ml, Sigma, Munich, Germany) for 10 min at7°C to enhance the penetration of the antibodies, washedwice in Tris-buffered saline (TBS) and blocked with
vidin/Biotin Blocking Kit according to manufacturer’s 1
rotocol (Vector Laboratories). The washed slides werencubated either with a polyclonal anti-TF antibody (1,12)pAb-sTF, 6 �g/ml) or a monoclonal anti-desmin antibodyclone D33, DakoCytomation, 2.5 �g/ml) overnight at 4°C;egative controls were performed without the use of primaryntibodies. The detection of primary antibodies was per-ormed as described earlier. To ensure that the counterstain-ng with hematoxylin had no influence on the TF staining,ontrol staining was performed without the use of hema-oxylin.
estern blot. Samples from explanted heart tissues wereomogenized in radio-immunoprecipitation assay-bufferupplemented with 1% protease-inhibitor cocktail (P-8340,igma). For immunoprecipitation extracts were incubatedvernight at 4°C with a monoclonal antibody anti-TFMabTFH clone TFE, Enzyme Research Laboratories,outh Bend, Indiana) and afterwards precipitated withrotein-G Plus Agarose (Santa Cruz Biotechnology, Santaruz, California). The precipitates were separated by
lectrophoresis on a 12% sodium dodecyl sulfate-olyacrylamide gel (SDS-PAGE). The gels were trans-erred to a nitrocellulose membrane (Protran BA 85,chleicher and Schüell, Dassel, Germany), which waslocked in 5% nonfat dry milk diluted in Tris-bufferedaline. Blots were incubated for 3 h at 4°C with the primaryolyclonal antibody anti-TF (pAb-sTF, 6 �g/ml). Washedlots were incubated for 90 min with a secondary horse-adish peroxidase-conjugated antibody (DakoCytomation).
ashed blots were subjected to the Immun-Star HRPhemiluminescent Kit (BioRad, Hercules, California) foretection of the immunoreactive signal by chemilumines-ence, and the membranes were then exposed to X-ray filmKodak X-OMAT AR, Kodak, Stuttgart, Germany). Theembranes were stripped in 100 mM glycine (pH 2.9) at
oom temperature for 60 min. The stripped blots werelocked again for 3 h and incubated with a primaryolyclonal antibody anti-asHTF (dilution 1:100, Pinedantikörper-Service). After the incubation, the blots wereashed and treated as described. The membrane was
xposed to X-ray film.rotein extraction and TF ELISA. Myocardial biopsiesere homogenized in a glass potter containing 100 �l
ce-cold radio-immunoprecipitation assay buffer, supple-ented with 1% protease-inhibitor cocktail, incubated on
ce for 30 min and centrifuged at 10,000 g for 10 min at°C. Total protein content of the cell lysate was measuredsing a bicinchoninic acid protein assay according to theanufacturer’s protocol (Pierce, Rockford, Illinois). The
rotein was precipitated with acetone and afterwards resus-ended in TBS, pH 8.5. To quantify the total TF proteinontent (full-length TF and asHTF) in myocardial biopsies,F ELISA was performed according to the instructionanual (Imubind Tissue Factor ELISA Kit, Americaniagnostica Inc.).
tatistical analysis. SPSS statistical software version
1.0.1 was used for statistical analysis. Nominal data were
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rdDmccoadsspcpqPs
1084 Szotowski et al. JACC Vol. 45, No. 7, 2005Altered TF Expression in Cardiomyopathy April 5, 2005:1081–9
ested using chi-square and Fisher exact test. Tissue factorRNA ratios obtained by real-time PCR were presented asean � SD. If more than two groups were compared,etric normally distributed data were analyzed by one-way
nalysis of variance. If this test revealed significant differ-nces, a Bonferroni-corrected (correction factor 3) non-aired t test was performed. Other metric normally distrib-ted data were expressed as mean � SD and compared byonpaired t test. A p value �0.05 was regarded asignificant.
ESULTS
linical characteristics of patients and hemodynamicarameters. The patients, from whom myocardial biopsiesere obtained, were divided into two groups according to
he EF (EF �50% [n � 37] or EF �50% [n � 42]). Theseroups were then subdivided into another two groupsepending on whether mRNA or protein levels were deter-ined. The explanted heart tissues from DCM patientsere assessed as a separate group (n � 12). The leftentricular end-diastolic diameter was significantly in-
Table 2. Clinical Characteristics of the PatientHeart Donors (Patients With DCM and SeverWith Moderate to Severely Reduced EF (�50(�50%, n � 19)
*p � 0.001 (explanted vs. EF �50%); †p � 0.01 (EF �50%(EF �50% vs. EF �50%).
ACE � angiotensin-converting enzyme; DCM � dilate
reased in the DCM patients compared to those patients a
ith normal EF (Tables 2 and 3). There was no significantifference in cardiovascular risk factor profile between thebove groups (Tables 2 and 3).
A statistical difference between the biopsied patients withegard to the medication with beta-blocker, diuretics, andigitalis was found as shown in Tables 2 and 3.etection of asHTF mRNA and protein in humanyocardial tissue. AsHTF mRNA was identified in myo-
ardial biopsies and explanted hearts from DCM patients byonventional RT-PCR. The gel electrophoresis showed notnly the PCR product for full length TF but also anmplification product for asHTF (Table 1A, Fig. 1). Theirect sequencing of the PCR products revealed specificequences for full-length TF and asHTF. The asHTFequence and protein were previously described to beresent in human leukocytic cells but not in human myo-ardial tissue (10,13). This is the first report about theresence of asHTF in human adult myocardial tissues. Theuantification of TF and asHTF mRNA levels by real-timeCR in myocardial biopsies taken from the right ventriculareptum of patients with a normal EF revealed myocardial
luded for TF Gene Expression Studies:educed EF, n � 12) and Biopsied Patients� 27) and Mildly Reduced to Normal EF
�50%); ‡p � 0.05 (EF �50% vs. EF �50%); §p � 0.001
omyopathy; EF � ejection fraction; TF � tissue factor.
s Incely R%, n
xpla
vs. EF
d cardi
sHTF expression level to be significantly lower than
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1085JACC Vol. 45, No. 7, 2005 Szotowski et al.April 5, 2005:1081–9 Altered TF Expression in Cardiomyopathy
yocardial TF mRNA level (13.91 � 10�5 � 11.20 �0�5 for asHTF/GAPDH ratio vs. 1.76 � 10�1 � 6.08 �0�2 for TF/GAPDH ratio; p � 0.0001).To find out whether asHTF protein was expressed in theyocardium, western blot analysis was performed. Full
ength TF and asHTF were both detected by the polyclonalnti-TF antibody (pAb-sTF, 6 �g/ml) directed against thextracellular domain of the TF protein (Fig. 2, lane 1).ehybridization of the membrane with the asHTF-specific
ntibody yielded a single protein band at 30 kDa, whereasull-length TF was not detected (Fig. 2, lane 2).
Immunohistochemistry showed cardiomyocytes and en-othelial cells to be positive for asHTF in myocardialiopsies of patients with normal EF and DCM. However,n contrast to full-length TF, which is abundantly expressedn the cardiomyocytes, asHTF-associated cardiomyocytetaining appeared to be faint (Figs. 3A and 3B). There waso difference in asHTF myocardial staining pattern com-aring patients with normal EF and with DCM (data not
able 3. Characteristics of Patients From Whom Biopsies Haveeen Taken for TF Protein: Biopsied Patients With Moderately
o Severely Reduced EF (�50%, n � 15) and Mildly Reducedo Normal EF (�50%, n � 18)
p � 0.001 (EF �50% vs. EF �50%); †p � 0.0001 (EF �50% vs. EF �50%);p � 0.003 (EF �50% vs. EF �50%); §p � 0.004 (EF �50% vs. EF �50%).
Abbreviations as in Table 2.
hown).b(
own-regulation of TF and asHTF messenger RNAxpression in DCM: correlation with the EF. Tissueactor isoform specific real-time PCR was performed touantify the expression levels of TF and asHTF mRNA inatients with normal EF and with DCM. The expression
evels of TF and asHTF were lower in DCM patients thann those with normal EF. Mean and standard deviation ofF/GAPDH and asHTF/GAPDH ratios are shown inigure 4. The myocardial TF expression in the biopsiedCM patients decreased to 60.2% (TF/GAPDH ratio 1.7610�1 � 6.08 � 10�2 in patients with normal EF vs. 1.06
igure 1. Gel electrophoresis (1% agarose gel) of reverse transcription-olymerase chain reaction products (human tissue factor [hTF] andlternatively spliced human tissue factor [asHTF]) using ribonucleic acidrom human heart biopsies. (M) 250 bp deoxyribonucleic acid ladder; (C)o template, control; (1) explanted heart tissue (ventricular septum); (2)uman myocardial biopsy from patient with normal cardiac function; (3)uman myocardial biopsy from patient with severely impaired cardiacunction.
igure 2. Western blot for tissue factor isoforms from explanted dilatedardiomyopathy hearts. Extracts from explanted dilated cardiomyopathyearts were immunoprecipitated with anti-human tissue factor (TF)ntibodies directed against the TF extracellular domain. Western blotshowed both, full-length TF and alternatively spliced human tissue factorasHTF) (lane 1), detected by a polyclonal anti-TF antibody (pAb-sTF)irected against the extracellular domain of the TF protein. Rehybridiza-ion of the membrane with a polyclonal antibody (asHTF), directed againsthe last 29 amino acids of the unique asHTF end yielded a single protein
and at approximately 30 kDa, whereas full length TF was not detectedlane 2).
�0DrEpph5141Ttwdwb0asDtwcd
FsaCopmpiip6dZstm6hTlocated around vacuoles (arrowheads). Original magnification 630�.
Fsor(mG
1086 Szotowski et al. JACC Vol. 45, No. 7, 2005Altered TF Expression in Cardiomyopathy April 5, 2005:1081–9
10�1 � 5.26 � 10�2 in biopsied DCM patients; p �.001). The myocardial asHTF expression in biopsiedCM patients decreased also to 51.5% (asHTF/GAPDH
atio 13.91 � 10�5 � 11.2 � 10�5 in patients with normalF vs. 7.17 � 10�5 � 3.82 � 10�5 in biopsied DCMatients; p � 0.014). Compared with the level present inatients with normal EF, TF mRNA levels in explantedeart tissues from DCM patients were also decreased to0.6% for TF (TF/GAPDH ratio 1.76 � 10�1 � 6.08 �0�2 vs. 8.91 � 10�2 � 2.95 � 10�2; p � 0.0001) and to2.1% for asHTF (asHTF/GAPDH ratio 13.91 � 10�5 �1.20 � 10�5 vs. 5.86 � 10�5 � 2.93 � 10�5; p � 0.014).he TF/GAPDH ratio and EF were positively correlated in
he patients included (r � 0.504; p � 0.0001; Fig. 5),hereas the TF/GAPDH ratio and left ventricular end-iastolic diameter (LVEDD) were negatively and onlyeakly correlated (r � �0.309; p � 0.018). Correlationsetween the asHTF/GAPDH ratio and EF (r � 0.382; p �.003; Fig. 5) as well as between asHTF/GAPDH rationd LVEDD (r � �0.278; p � 0.04) were also weaklyignificant.
own-regulation and cellular redistribution of TF pro-ein in the myocardium of patients with DCM. In lineith the mRNA results, protein quantification of total TF
ontent (full-length TF and asHTF) by ELISA revealed a
igure 4. Quantification of tissue factor (TF) expression in ventriculareptum of explanted dilated cardiomyopathy (DCM) hearts (n � 12) andf biopsied patients with DCM (n � 27) and with normal to mildlyeduced ejection fraction (EF) (n � 19). Data are given as mean � SD.A) Full-length TF/glyceraldehyde-3-phosphate dehydrogenase (GAPDH)essenger ribonucleic acid (mRNA) ratio; (B) Alternatively spliced TF/APDH mRNA ratio. asHTF � alternatively spliced human tissue factor.
igure 3. Immunohistochemistry for tissue factor (TF), alternativelypliced human tissue factor (asHTF), and desmin. Polyclonal rabbitntibodies directed against the extracellular domain of TF and the unique-terminal end of asHTF were used for staining of the myocardial biopsiesbtained from a patient with normal ejection fraction (EF) (64%) and aatient with dilated cardiomyopathy (DCM) (21%). (A and B) Cardio-yocytes and endothelial cells within the normal myocardium stained
ositive for asHTF. Original magnification 400�. (C) Immunohistochem-stry for desmin showed positive staining of Z-bands of the cardiomyocytesn a patient with normal EF. The sarcolemma and Z-bands of the sameatient were also positive for TF (arrows, D). Original magnification30�. (E) The Z-bands stained also positive for desmin in the myocar-ium of a patient with DCM. In contrary to desmin, TF staining of the-bands was faint or completely absent in the myocardial biopsy of the
ame DCM patient (arrows, F and G). Myocardial TF was located withinhe cytosol around the nucleus in DCM (arrow heads, F and G). Originalagnification 630�. (H) Control for TF staining. Original magnification
30�. (I) TF staining of the Z-bands of a patient with normal EF withoutematoxylin. Original magnification 630�. (J) Immunohistochemistry forF showed a diffuse staining with absence of TF-positive Z-bands. TF was
own-regulation of TF in the myocardium from patients
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1087JACC Vol. 45, No. 7, 2005 Szotowski et al.April 5, 2005:1081–9 Altered TF Expression in Cardiomyopathy
ith normal to mildly impaired EF compared with thoseaving moderate to severely impaired EF (8.8 � 3.0 ng/mgotal protein (n � 18) vs. 6.5 � 2.7 ng/mg (n � 15); p �.032; Fig. 6). Thus, the total myocardial TF proteinxpression was reduced by approximately 26%. MyocardialF protein correlated with the EF in biopsied patients
Fig. 6B).Immunohistochemical staining of adjacent specimens
rom myocardial biopsies with desmin and TF antibodiesevealed desmin and TF to be present in the Z-bands of theyocardium in patients with normal EF (Figs. 3C and 3D).issue factor was also found in the sarcolemma and inter-
alated disks of the myocardial tissue. In contrast to desminnd TF in hearts with normal EF, TF staining of the-bands was faint or completely absent in DCM (Figs. 3F
nd 3G). However, the Z-bands stained positive for desminn myocardial biopsies of the same patients with DCMFig. 3E). In the myocardial biopsies from DCM patients,F was localized in the perinuclear cytosol of the cardio-yocytes (Figs. 3F and 3G). Thus, the myocardial speci-ens from DCM hearts showed an altered cellular local-
zation of TF, redistributing from the Z-bands to theerinuclear cytosol in DCM. To ensure that the counter-taining with hematoxylin had no influence on the TFtaining, control staining was performed without the use ofematoxylin (Figs. 3I and 3J).Immunohistochemistry with and without hematoxylin
igure 5. Graphs demonstrate correlations between tissue factor (TF)soform expression and ejection fraction (EF). (A) Regression line betweenhe messenger ribonucleic acid (mRNA) TF/glyceraldehyde-3-phosphateehydrogenase (GAPDH) ratio and EF (r � 0.504, p � 0.0001). (B)egression line between the mRNA alternatively spliced human tissue
actor (asHTF)/GAPDH ratio and EF (r � 0.382, p � 0.003).
ounterstaining showed both TF staining of Z-bands in i
atients with normal EF (Figs. 3D and 3I), whereas anltered staining pattern without TF-positive Z-bands wasound in DCM (Figs. 3E and 3J).
ISCUSSION
his study shows TF expression to be down-regulated inhe myocardium of patients with DCM. Tissue factor haseen reported to be expressed by cardiomyocytes in thearcolemma and in the transverse part of the intercalatedisks, where it co-localizes with the cytoskeletal proteinsesmin and vinculin (4,5). The observation that TF expres-ion correlated with the number of contact sites of cardio-yocytes led to the hypothesis that TF might play an
mportant role in maintaining the structural integrity of theyocardial muscle. The cytoplasmic domain of TF was
hown to interact with actin-binding protein 280 (filamin),hereby influencing the adhesion and migration of vascularells (6). During postnatal maturation the increased degreef TF immunostaining present in the intercalated disksorrelates positively with the formation of intercardiomyo-yte desmosomal and junctional contacts and has beenuggested to possibly influence the electromechanical func-ion of the myocardial muscle (5,15). Lower TF levels haveeen reported in structurally altered ventricular myocardiumf patients with hypertension and ventricular hypertrophy.issue factor protein content was decreased approximately0% in tissue extracts from the myocardium of severelyypertrophic hearts (4). Here, we demonstrate the TFRNA level in the ventricular septum of patients withCM to be down-regulated to approximately 60% of that
igure 6. (A) Quantification of tissue factor (TF) protein (ng TF pro-ein/mg total protein) from biopsied patients with dilated cardiomyopathyn � 15) and normal to mildly reduced ejection fraction (EF) (n � 18).ata are given as mean � SD. (B) Correlation between TF protein and EF
r � 0.423, p � 0.014).
n patients with normal cardiac function. Tissue factor
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oAs
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R
1088 Szotowski et al. JACC Vol. 45, No. 7, 2005Altered TF Expression in Cardiomyopathy April 5, 2005:1081–9
rotein content in biopsied DCM patients was reduced bypproximately 26%. In addition to the reduction in TFxpression, cellular TF localization was altered, with aislocation of TF from the Z-bands to the cytosol of theardiomyocytes, suggesting that the reduction and redistri-ution of TF may contribute to myocardial dilation andontractile dysfunction. Whether down-regulation of TFxpression and its cellular translocation contributes to ven-ricular dilation and cardiac dysfunction needs to be furtherlucidated. Tissue factor interacts with tissue factor pathwaynhibitor, which is expressed on cardiomyocytes (16). Ofote, TF pathway inhibitor is known to bind heparin-ulfated proteoglycans on various cell types, thereby con-ributing to cell stabilization. A reduction in myocardial TFxpression is likely to produce an overall decrease in theumber of TF molecules localized, via its modified intra-ellular cysteine residue (17), to lipid rafts—membraneormations involved in regulation of actin cytoskeleton (18).
It was previously reported (19) that the spatial expressionf membrane-bound TF corresponded to that of force-ransmitting cytoskeletal proteins in actin-rich membranereas of in vitro cultured epithelial cells. In line with theo-localization of TF with other structural proteins is thending that the myocardial TF expression correlated withhe ejection fraction in our patients. Data from animalnockout experiments and clinical findings on patients withCM suggest that defects in cytoskeletal and junctional
roteins may cause cardiac dilation, contributing to theevelopment of cardiac pump failure (8). A reduced TFrotein content of the left ventricular myocardium has beenescribed for patients with sepsis. It has been suspected thathe reduced TF expression by cardiomyocytes may contrib-te to cardiac failure during sepsis (5). The reason for theeduction of TF expression during sepsis is thus far un-nown (20).Differences in medication with beta-blocker, diuretics,
nd digitalis were present between our patient groups. Moreatients with DCM were treated with these drugs thanatients with normal or mildly reduced EF (Tables 2 and 3).t is not known to date whether medication with beta-locker, diuretics, or digitalis influences TF gene expression.owever, clinical and experimental studies suggest that
tatins and angiotensin-converting enzyme (ACE) inhibi-ors reduce the TF expression in human monocytes (21,22)nd endothelial cells (23,24). No differences in the fre-uency of statin or ACE inhibitor administration werebserved between our patient groups. Thus, we can excludehat a difference in TF gene expression between our patientsroups is related to differences in therapy with statins orCE. Nevertheless, it should be emphasized that theechanisms and regulation pathways of TF gene expression
n cardiomyocytes are poorly understood.Here we also report the presence of asHTF in cardiomy-
cytes and endothelial cells of the human myocardium.lternatively spliced human tissue factor has been demon-
trated to circulate in the blood of healthy human beings,
eing able to induce factor Xa generation (10). Acellular TFas also been reported to be present in blood of patientsndergoing coronary artery bypass grafting (25). In theseatients, the fluid-phase form of TF failed to supporthrombin generation, whereas full-length TF present inicroparticles in the blood promoted thrombin generation
25). Acellular TF may bind to TF pathway inhibitor,hereby modulating blood thrombogenicity. Leukocytesave been suggested as a source for asHTF in blood, ashese cells are able to synthesize asHTF (10). Whetherndothelial cells contribute to the presence of asHTF in thelood under pathologic conditions is unknown. In compar-son with full-length TF, only a limited amount of asHTFrotein was found to be present in the right ventriculareptum. The quantification of myocardial TF expressionhowed that both full-length TF and asHTF were reducedn patients with DCM. In addition, the expression ofull-length TF in myocardial tissues correlated well withyocardial contractility, pointing to an association of trans-embrane TF expression and myocardial function. A phys-
ological role for asHTF, which is a soluble protein, has thusar not been identified for the heart. In addition, theorrelation between the soluble isoform of TF and EF wasnly weak. Whether asHTF is expressed to a larger extentithin the myocard of the atrium and/or the reduction of
sHTF in DCM contributes to dysfunction of the myocar-ial muscle remains a subject of further investigation.In summary, the TF expression was down-regulated in
he myocardium of DCM patients. The reduction in TFxpression and its dislocation from the Z-bands into theytosol of the cardiomyocytes may adversely affect theell-to-cell contact stability, thereby contributing to ventric-lar dilation and cardiac dysfunction in DCM.
cknowledgmentshe authors acknowledge Prof. Dr. Körfer, Director, Clinicf Thoracic and Cardiovascular Surgery, Herzzentrumordrhein-Westfalen, Bad Oeynhausen, Germany, for pro-
iding some of the explanted DCM hearts. The authors thankr. S. Schwartz, Department of Haematology, Charité-niversitätsmedizin Berlin, Germany, for technical support on
ight microscopy and digital photography, and Michael Pi-rkowski for support in the manuscript preparation.
eprint requests and correspondence: Dr. Ursula Rauch, Medicallinic II, Charité-Universitätsmedizin Berlin, Campus Benjaminranklin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail:[email protected].
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