BRC 01 003451
Electronic e* appendix:
Table 1:
Clinical and hemodynamic data in patients studied. AS: aortic stenosis, DCM dilated cardiomyopathy, LV = left ventricular, EDD enddiastolic dimension, ESD endsystolic dimension, EDP enddiastolic pressure, d p max and mean: maximal and mean pressure gradient at aortic valve, ACEI: ACE inhibitors. NYHA (numbers of patients in classes II/III/IV) are indicated.
Clinical features in groups AS I and AS II, respect. DCM I and DCM II, are not different.
AS (1+II) and DCM (1+II) are different in all parameters except sex, as to be expected between patients undergoing valve surgery and end stage heart failure.
PCR Primers and real time PCR
NEP primers: (forward: 5`-GAA CCTACA AGG AGT CCA GA-3` and reverse: 5`-GCT CCA CTT ATC CAC TCA TC-3` and GAPDH (forward: 5`-CAC CAT CTT CCA GGA GCG AG-3`, reverse: 5`-GCA GGA GGC ATT GCT GAT-3`)). In the Light CyclerTM , the specifity of the reaction was evaluated by the appearance of a single-peak melting curve. SYBR Green signals from each probe were related to a standard curve in each assay (50ng, 25ng, 12.5ng, 6.25ng and 3.125ng of total RNA from pooled samples).
NEP activity determination, details:
Myocardial tissue samples (8 - 24 mg) were homogenized using a glass-on-glass potter homogenizer (20 strokes, 4 °C) in 20 parts (v/w) Tris/HCl buffer (0.1 M, pH 7.6) containing 1% Triton X-100, 0.5 mg/ml soybean trypsin inhibitor and 200 IU/ml aprotinin. After removal of crude debris by centrifugation (1500 xg, 5 min), supernatant was incubated with 0.6 mM Suc-Ala-Ala-Phe-AMC for 30 min at 37°C in the absence and presence of the NEP-inhibitor thiorphan (2.5 µM). The reaction was stopped with Na-EDTA (2 mM) and thiorphan (2.5 µM), and proteins were removed by heat precipitation (10 min, 95°C). 7-amino-4-methyl-coumarin (AMC) was released from the reaction product Phe-AMC by a subsequent incubation with aminopeptidase M (0.8 IU/ml, 5 min, 37 °C), and was quantified by fluorometry (360/440 nm). The thiorphan-sensitive reaction was considered to represent NEP-activity. Proteins were measured by the method of Lowry et al.
In situ PCR
The paraffin-embedded heart muscle specimens were cut in 5-µm sections, mounted on 3-aminopropyl-triethoxysilane-coated glass slides (Perkin–Elmer, USA), and were incubated 2 hours at 60 °C in an incubator. The sections were dewaxed by washing twice in xylol, first for 20 min at 37 °C, then 20 min at room temperature. Residual xylol was extracted by placing the samples in 80, 90, and 100% ethanol for 3 min each. Tissue was air-dried. The slides were washed with HCl 1x PBS and Triton X 100 for permeabilization, followed by proteinase K digestion (30 min, 37 °C). Then the acetylation step was performed with acetic anhydride in triethanolamine followed by acetic acid treatment and washing in distilled water. Then again dehydration was performed in a series of graded ethanols.
For reverse transcription thermolabile viral reverse transcriptase (MMLV, Gibco, Germany) was used (30 min, 37 °C, and 15 s, 65 °C). Following the reverse transcription the reaction components were washed of the slides and the tissue was fixed briefly to maintain the localisation of the cDNA in situ.
PCR amplification was conducted on an in-situ thermal cycler (Perkin–Elmer). We used labeled primers instead of labeled nucleotides to avoid background activity resulting from unspecific repair mechanisms of taq polymerase. Primers were labeled with three DNP-groups to enhance signal activity. The PCR mix was trapped over the air-dried samples by silicon diaphragms using mounting clips and an assembly tool (Perkin–Elmer). With a standard in-situ amplification mixture (Perkin Elmer) the following PCR protocol was performed: 70 °C preheating, hot start (5 min, 94 °C), 36 cycles (1,5 min, 55 °C, and 30 s, 94 °C). Following PCR, slides were washed and each section was covered with 45 ml of alkaline phosphatase anti DNP antibody solution (1:500 in casein) and was incubated at room temperature for 30min. After a brief wash in PBS the enzyme was reacted with the substrate 5-bromo-4-chloro-3-indolyl phosphate in the presence of nitrobluetetrazolium to produce a water insoluble purple black formazan dye at the site of the amplicon. Fotographs were taken with a Nikon Coolpix 900 digital camera with a Zeiss Axiolab microscope.
For each tissue sample, 3 controls were performed in which homologous sections were treated with a reaction mixture without dNTP, taq-polymerase or primers, respectively. Only when controls were tested negative, a positive reaction was accepted. [1]
1. Morrison, C., et al., In situ determination of B-cell heavy chain and kappa/lambda light chain expression patterns: methodology and clinical utility. Diagn Mol Pathol, 2001. 10: 171-8.
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Table 1
group / n / Age[years] / Sex
[% male] / NYHA
(II/III/IV) / LVEDD
[mm] / LVESD
[mm] / LVEF
[%] / LVEDP
[mmHg] / Dp max
[mmHg] / Dp mean
[mmHg] / ACEI
[n]
AS-I / 19 / 60±3.3 / 69 / (8/11/0) / 52±2.6 / 36±3.6 / 52±3.5 / 17±2.0 / 76±7.0 / 48±5.7 / 5
AS-II / 8 / 70±3.4 / 80 / (3/5/0) / 54±3.7 / 42±5.1 / 45±7.6 / 18±3.2 / 68±10 / 42±6.6 / 4
DCM I / 14 / 44±4 / 100 / (0/5/9) / 73±3.1 / 63±3.8 / 25±4.0 / 24±3.3 / 14
DCM II / 8 / 41±3 / 100 / (0/3/5) / 79±3.2 / 71±3.5 / 20±3.6 / 23±2.4 / 8
p (AS I+II) vs
(DCM I+II) / <0.0001 / 0.168 / <0.0001 / <0.0001 / <0.0001 / <0.0001 / 0.03
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