Supplementary Online Information s1

MS #1460/R2 1

Supplementary Online Information Methods Real-time PCR Analysis Primers for real time PCR were designed using Prime software (Genetics Computer Group Package, Wisconsin, USA). For sequences of oligonucleotides used, see Table 1. PCR was performed using “Hot Start” reaction mix (Light Cycler – FastStart DNA Master SYBR Green I). cDNA (1 µl of the RT-PCR reaction) was added to a 20-µl RT-PCR reaction mixture according to the manufacturer's protocol. The temperature profile included an initial denaturation for 10 min at 95° C, followed by 37 cycles of denaturation at 95° C for 15 sec, annealing at different temperatures (Table 1 online) for 5 sec, and elongation with fluorescence monitoring at 72° C for 40 sec. LCDA Version 3.1.102 was used for PCR data analysis. The specificity of the amplification reaction was determined by performing a melting curve analysis. Standard curves for expression of each gene were generated by serial dilution of known quantities of the respective cDNA gene template. Relative quantification of the signals was done by normalizing the signals of the different genes with the ß-actin signal.

Table 1 online. Primer sequences and annealing temperatures used cDNA Primer Sequence Annealing Temp. and Primer VEGF 165 sense 5´-AGCCTTGCCTTGCTGCTCTA-3´ 58° C antisense 5´-GTGCTGGCCTTGGTGAGG-3´ VEGF 189 sense 5´-AGCCTTGCCTTGCTGCTCTA-3´ 60° C antisense 5´-GGGATTTCTTGCGCTTTCG-3´ VEGF-B sense 5´-TGCAGATCCTCATGATCCG-3´ 60° C antisense 5´-TGTCTGGCTTCACAGCACTG-3´ VEGF-C sense 5´-CCCCAAACCAGTAACAATC-3 58° C antisense 5´-CACAGGCACATTTTCCAG-3´ ß-Actin sense 5´-TGCCATCCTAAAAGCCAC-3´ 58° C antisense 5´-TCAACTGGTCTCAAGTCAGTG-3´ MS #1460/R2 2

Western Blotting Biopsies were lyzed in solubilization buffer (10 mM Tris-Cl, 50 mM NaCl, 1% Triton X-100,

30 mM sodium pyrophosphate, 100 µM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1x CompleteTM - EDTA-free Protease Inhibitor Cocktail (Roche, Mannheim, Germany). Insoluble material was removed by centrifugation (15,000 rpm, 30 min, 4° C). Tissue lysates (50 µg/lane) were separated by 7.5% or 10% SDS-PAGE prior to electrophoretic transfer onto Hybond C super (Amersham Pharmacia Biotech, Buckinghamshire, UK). Proteins were immunodetected on the membrane using chemiluminescence (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK) and specific protein bands were quantified using Easy plus Win 32 software (Herolab, Wiesloch, Germany).

From each heart two cardiac biopsies were prepared for TEM analyses and embedded in Epon. From each biopsy, four ultra thin sections were made and analyzed in TEM at 1000x magnification. The grid was divided in 4 quadrants; and the upper right and the lower left quadrant (resulting in an equal morphometry area) were used. In each quadrant two fields were used, yielding 32 fields for each heart, and a total of 672 fields for DCM, 640 fields for ICM and 320 fields for controls. In each field 60 mitochondria were classified as normal, edematous, and degenerated as described earlier 1. Then, images were obtained from each field and transferred into the computer and analyzed using a morphometry software (Lucia, Nikon Instruments, Vienna, Austria). Capillaries in each field were identified and marked with white points. The border of myocytes was marked with thick white lines and the border of collagen was marked with thin white lines. Binary images were produced and following parameters were evaluated for each field automatically: 1. The mean number of capillaries; 2. The average area occupied by myocytes; 3. The average area occupied by collagen. Then the data were pooled for ICM, DCM, and controls and mean values were calculated.

Data Analysis

The mRNA and protein expression levels of all molecules were compared between normal and CMP cardiac biopsies, based on the diagnosis DCM or ICM. In a next step, we compared the number of capillaries and morphometric data on myocytes and collagen between the two MS #1460/R2 3 patient groups and non-failing cardiac biopsy. Analysis of variance was used for statistical analysis. Data are expressed as mean values and statistical significance is set at p < 0.05.

References

1. Alessandrini F, Thakkar M, Foda HD, Said SI, Lodi R, Pakbaz H, Schraufnagel DE. Vasoactive intestinal peptide enhances lung preservation. Transplantation. 1993; 56:946-73.