Online Appendix for the following February 22, 2011 JACC article
TITLE: Diastolic Tissue Doppler Indices Correlate With the Degree of Collagen Expression and
Cross-Linking in Heart Failure and Normal Ejection Fraction
AUTHORS: Mario Kasner, MD, MSc, Dirk Westermann, MD, Begoña Lopez, DSc, Regina
Gaub, MD, Felicitas Escher, MD, Uwe Kühl, MD, Heinz-Peter Schultheiss, MD, Carsten
Tschöpe, MD
APPENDIX
Supplementary Methods
Histology and Immunhistology
Formalin-fixed tissue was processed for paraffin embedding, sectioned at standard thickness,
and stained with collagen-specific picrosirius red (F3BA in aqueous picric acid)(6). The fraction
of myocardial volume occupied by fibrillar collagen (collagen volume fraction or CVF) was
determined by quantitative morphometry with an automated image analysis system (AnalySYS,
Soft-Imaging-System GmbH, Hammer) (6). To distinguish between cross-linked (insoluble) and
noncross-linked (soluble) collagen, a colorimetric procedure was used(38). First, a fast greensirius red assay was performed to identify and quantify total collagen. In a second step, a sircolbased assay was performed to obtain and quantify soluble collagen. The amount of insoluble
collagen was calculated by subtracting the amount of soluble collagen to the amount of total
collagen. The degree of cross-linking was calculated as the ratio between the insoluble and the
soluble forms of collagen. Cross-linking was determined retrospectively in only 23 of 41 patients
(14 HFNEF and 9 controls) where EMBs samples were available.
To determine collagen type I and III, immunohistological stainings were performed using
standard techniques. The antibodies were purchased from Chemicon (anti-collagen type-I,
1:225 and type-III, 1:75). Immunohistochemical stainings were quantified by digital image
analysis (DIA)(39). Basically, DIA macros consisted of: 1)grabbing of the image; 2)recognition of
artifacts for the calculation of the net myocardial area (Heart Area/HA= area of measurement
frame – lit area/mm2); 3) recognition of stained collagen (red, yellow and green) under polarized
light. The data evaluated by these LUCIA macros (LUCIA-Gv.3.52ab, Nikon GmbH,Germany)
were transferred to a Microsoft Excel data processing macro, which calculated the net 'Heart
Area' (HA) and the means of measured qualities (Area Fraction=AF).
In addition, the expression of lysyl oxidase (LOX), the enzyme responsible for collagen
cross-linking was determined retrospectively in 15 patients where EMBs samples were available
(10 HFNEF and 5 controls). Immunhistochemical analysis for LOX was performed on formalinfixed and paraffin-embedded sections, and staining was performed by the avidin-peroxidaselabeled dextran polymer method. Positive staining was visualized with DABPlus (Boehringer
Mannheim Corp.), and tissues were counterstained with Harris hematoxylin (Sigma). A mouse
monoclonal antibody against LOX (R&D Systems) was used as the primary antibody. The
results are expressed as semiquantitative values (absent, mild, moderate and intense
expression).
The histomorphological study was performed by a pathologist blinded for patient data.
To determine myocyte dimensions the maximal myocyte diameter was measured through the
nucleus from 10 cells per sample. Using standard equation for sample size, 10 measurements
reduced the sampling error to <3%.
RNA isolation, reverse transcription
Total RNA was extracted from the EMB by the Trizol-method (GIBCO BRL, Carlsbad,
California). Additional purification was performed using the ChargeSwitch® Total RNA Cell-Kit
(Invitrogen, Karlsruhe,Germany). The yield of purified total RNA was analyzed by checking the
UV absorbance at 260 nm on the NanoDrop® ND-1000 spectrophotometer (Agilent
Technologies, Boeblingen,Germany). Reverse transcription from 30µl of total RNA was carried
out using the High Capacity cDNA Archive-Kit (Applied Biosystems/ABI, Darmstadt, Germany)
to a final volume of 60µl according to the manufacturer’s instructions.
Preamplification and RT-PCR
The ABI inventory TaqMan® gene expression assays (including forward and reverse primers,
and fluorescently marked probe) are used for preamplification and for the real-time-PCR (RTPCR) according to the manufacturers’ protocol. Prior to RT-PCR, 1-250ng of cDNA samples
was preamplified with the pooled gene expressions assays using TaqMan® PreAmp Master Mix
(Early access) in a final volume of 25µl. The PCR program consisted of 10min denaturation step
at 95°C following by 14 cycles each of 4min at 60°C and 15sec at 95°C. The preamplification
products were diluted 1:20 with undiluted TE-buffer (10mM Tris/HCl pH 7.5, 1mM EDTA). 2µl of
diluted cDNA were used for the RT-PCR. RT-PCR was performed with TaqMan® Universal
PCR Master Mix according to the manufacturers’ protocol. The PCR reactions prepared without
cDNA were transferred to a Thermo-Fast® 96 Detection Plate (ABgene, Hamburg,Germany)
and then 2µl of preamplified cDNA were added to each well. Plates were sealed with a
MicroAmp™ Optical Adhesive Film (ABI, Darmstadt, Germany). The PCR was run on an
ABI7900 HT Fast RT-PCR System (Applied Biosystems, Darmstadt, Germany) with the
following program: hot start at 50°C for 2min, denaturation at 95°C for 10min and 40 cycles of
amplification (95°C for 15sec and 60°C for 1min). Quantification of housekeeping CDKN1B
transcripts as an internal control for the amount and quality of cDNA was undertaken for all
samples.