Online Appendix for the following JACC article
TITLE: Cardiac Fibrosis in Human Transplanted Hearts Is Mainly Driven by Cells of
Intracardiac Origin
AUTHORS: Martin Pichler, MD, Peter P. Rainer, MD, Silvia Schauer, Gerald Hoefler, MD
APPENDIX
Supplementary data
Immunohistochemical analysis of collagen type I, type III and CD68
After deparaffination, immunostaining with antibodies specific to collagen type I (Monosan, Cat. No.
PS 047, Vienna, Austria), collagen type III (Monosan, Cat. No. PS049, Vienna, Austria) and CD68
(Dako, Cat. No. M0814, Vienna, Austria) was carried out using an indirect streptavidin–biotin method
on a Dako autostainer.
CISH analysis
Three samples from gender-matched male heart transplants and two samples from female heart
biopsies served as positive and negative controls, respectively. To control for inconsistencies of
masking antigen and hybridization sites by different storage durations of our biopsies we selected
positive controls that had been stored 1 yr, 5 yrs and 15 yrs, respectively. The advantage of using
small pieces of endomyocardial biopsies (instead of whole organs) lies in the short diffusion
distance and therefore complete and rather homogenous penetration of the formalin fixative.
The controls were treated exactly the same way as the samples of interest and we used
controls with different storage times (1 year, 5 years and 15 years after harvesting). To test the
sensitivity of CISH staining we counted Y-chromosome specific signals in 5,000 nuclei of
1
cardiomyocytes, in 2,000 spindle to ovoid-shaped nuclei of putative fibroblast in areas of increased
fibrosis, and in 200 nuclei of CD68-positive stained macrophages. Four-micrometer thick sections
were prepared from formalin-fixed paraffin-embedded (FFPE) right ventricular endomyocardial
biopsies. CISH analysis was performed using the Spot-Light Y Chromosome Probe kit (Zymed
Laboratories Inc., California, US) according to the manufacturer’s recommendations. Finally, the
slides were counterstained with hematoxylin and covered with a Histomount™ Mounting Solution
(Zymed Laboratories Inc., California, US). As macrophages can display “spindle-shaped” cell nuclei
imitating fibroblasts, which could lead to misinterpretation of the results, we utilized a second strategy
by combining CISH analysis with a CD68 immunohistochemical staining procedure. To identify
macrophages, we added a CD68-specific antibody (Dako, Cat. No. M 0814, Vienna, Austria) before
counterstaining with hematoxylin and performed indirect AEC staining (Sigma-Aldrich, Schnelldorf,
Germany). The hybridization efficiency and specificity were determined in cardiac biopsies of nonmismatched transplanted male and female patients. No positive signals were found in negative control
female hearts, suggesting high specificity of the CISH analysis. Among the three male gender-
matched positive controls, 50±10% of the nuclei of cardiomyocytes were positive for the Ychromosome. In areas of increased fibrosis 87±5% of the spindle to ovoid-shaped nuclei of
the putative fibroblasts were positive for the Y-chromosome and 90±7% of the nuclei of the
CD68-positive macrophages. For sensitivity testing in cardiomyocytes, we counted 5,000
cardiomyocytic nuclei for each of the three positive controls and found a sensitivity of 50%
(±10) regarding Y chromosome positive cells in cardiac tissue samples of male patients. The
number of 5,000 counted nuclei is comparable to (or even greater than) some previously
published chimerism studies (e.g.(1,2)), and the sensitivity of 50% is also in the range of
previously published studies (e.g., (3,4))
DNA isolation and genotyping of type III collagen
We determined the presence of both SNPs in the DNA from seven donor hearts and in
material of their corresponding originally explanted recipient hearts. For this purpose,
2
genomic DNA from endomyocardial biopsies was extracted using a standard protocol for
DNA extraction from FFPE tissue. The samples were then evaluated by PCR. For each PCR
reaction, genomic DNA was used as a template for amplification of a 294-bp fragment of
exon 30 and a 326-bp fragment of exon 32. PCR was conducted in a standard thermocycler
using a final reaction volume of 50 µl that contained 100 ng genomic DNA template, 1 x PCR
buffer, 0.1 mM dNTPs, 25 pmol of each primer (exon 30: 5´AAGTATACAAATTTCTAGATTG-3´, 5´-ATAAATGATCAGAAGGAAATCA-3´, exon
32: 5´-CAACACTCCTGGAAAGTAATCG-3´, 5´-AGTGCAGGACTGTCCCATATG-3´)
and 0.5 U “Hot Star” Taq polymerase (Qiagen, Hilden, Germany). Reactions were started
with an initial 15 min AmpliTaqGold activation step at 95°C followed by 35 cycles at 94°C
for 1 min, 52°C for 1 min and 72°C for 2 min. For RFLP analysis of exon 30, 3 µl of the PCR
product was mixed with 20 U of the restriction enzyme AluI (New England Biolabs,
Frankfurt, Germany) and 2.5 µl 10 x NEB buffer in a total reaction volume of 20 µl. For
analysis of exon 32, 5 µl of the PCR product was mixed with 5 U of HaeIII (New England
Biolabs, Frankfurt, Germany). After an incubation step at 37°C for 1 hr, the digested
fragments were analyzed on a standard agarose gel, and allelotypes were determined
according to the protocol by Chen et al. (5).
RNA isolation and origin specific collagen type III expression
For this purpose, we had to redesign primers for exon 30 and exon 32 of the COL3A1 gene
using the freely available Primer3 program. This was necessary to shorten the length of the
amplicon because we had to use mRNA extracted from FFPE tissue that usually does not
exceed 120 bases in length. Total RNA was isolated using a TRIzol-based standard RNA extraction
protocol and finally treated with DNase (TURBO DNA-free, Ambion, Darmstadt, Germany) to avoid
genomic DNA contamination. Importantly, recovering RNA from formalin-fixed tissue can be
challenging. Our laboratory was involved in the European FP6 program IMPACTS
3
(www.impactsnetwork.eu, (6)), a multicenter research initiative for evaluation and
standardization of nucleic acid extraction from FFPE tissue. We applied stringent criteria for
the assessment of the quality and the results of RNA-based methods. After harvesting the
endomyocardial biopsies, they were fixed for 24 hours in 4% buffered formalin, paraffin
embedded and finally stored until RNA extraction. The time of storage ranged between 1 to
15 years. Controls to exclude genomic DNA contamination included for all samples a nonreverse transcript RNA sample and a no template control. Each reaction was performed in
duplicate and the presence of false positive (i.e. genomic contamination) and unspecific PCR
products was ruled out by analysis of PCR products on agarose gels. PCR was conducted in a
final reaction volume of 50 µl. Each reaction contained 3 µl cDNA template, 5 µl 10 x PCR
buffer,
0.1
mM
dNTPs,
25
pmol
of
each
primer
(exon
30:
5´-
CCTGGTGAACGTGGACCTCCTG-3´, 5´-TTCCTCCTTCGGGACCAGGG-3´, exon 32:
5´-CTTGGAAGTCCTGGTCCAAA-3´, 5´-AGGACCAGTAGGACCCCTTG-3´), and 0.5 U
“Hot Star” Taq polymerase (Qiagen, Hilden, Germany). The amplified PCR products were
pretreated for the primer extension reaction with 2 U exonuclease I (New England Biolabs,
Frankfurt, Germany) and 1 U shrimp alkaline phosphatase (Roche, Mannheim, Germany) for
45 min at 37°C followed by 15 min at 85°C. The primer extension reaction was performed in
a total volume of 10 µl, containing 5 µl pretreated PCR product, 0.5 to 5 pmol extension
primer
(exon
30:
5´-GGCCCCAGGACTTAGAGGTGGA-3´,
exon
32:
5´-
GACAAGGGTGAACCAGGCGG-3´) and 5 µl SNaPshot Multiplex Ready Reaction Mix
according to the manufacturer’s protocol. This reaction mixture was incubated at 95°C for an
initial step of 2 min followed by 25 cycles of 95°C for 5 s, 50°C for 5 s and 60°C for 5 s. A
final incubation with 0.5 U shrimp alkaline phosphatase for 1 hr at 37°C and enzyme
inactivation for 15 min at 75°C was performed to avoid fluorescent background by
unincorporated ddNTPs. Aliquots of 0.5 µl SNaPshot product, 0.5 µl of GeneScan 120 LIZ
size standard (Applied Biosystems, Vienna, Austria) and 9 µl Hi-Di formamide (Applied
4
Biosystems, Vienna, Austria) were mixed, heated to 95°C for 5 min to denature the DNA and
loaded on a 96-well microamp plate (Applied Biosystems, Vienna, Austria).
Sensitivity testing of the assay
For testing the sensitivity of the assay, we performed dilution experiments: pre-defined dilutions of
heterozygous and homozygous genomic DNA were mixed in one reaction tube. These experiments
showed that, depending upon the genotype, 0.5% to 5% of recipient-derived nucleic acids is
reproducibly quantifiable by this technique in the background of the dominating allele
(Table). We first tested the sensitivity of the assay by mixing two unrelated DNAs at a variety
of dilutions and determined that the marker SNPs could detect at least 5% recipient DNA in a
background of donor DNA. Our sensitivity data are in line with previously published
sensitivity limits reported for this technique of quantitative SNP allele frequency
measurement (7). By increasing the primer concentration from 0.5 to 5 pmol, we could lower
the detection limit to 0.1%. However, because the fluorescence peak of the dominating allele
reached the upper detection limit under these conditions, this measurement was qualitative
rather than quantitative (Figure S1). Because no product was detectable from RNA samples
before the reverse transcription step, contamination of genomic DNA could be excluded.
REFERENCES Supplementary Information
1.
2.
3.
4.
5.
6.
7.
5
Bayes-Genis A, Salido M, Sole Ristol F, et al. Host cell-derived cardiomyocytes in
sex-mismatch cardiac allografts. Cardiovasc Res 2002;56:404-10.
Muller P, Pfeiffer P, Koglin J, et al. Cardiomyocytes of noncardiac origin in
myocardial biopsies of human transplanted hearts. Circulation 2002;106:31-5.
Glaser R, Lu MM, Narula N, Epstein JA. Smooth muscle cells, but not myocytes, of
host origin in transplanted human hearts. Circulation 2002;106:17-9.
Rupp S, Koyanagi M, Iwasaki M, et al. Characterization of long-term endogenous
cardiac repair in children after heart transplantation. Eur Heart J 2008;29:1867-72.
Chen HY, Chung YW, Lin WY, Wang JC, Tsai FJ, Tsai CH. Collagen type 3 alpha 1
polymorphism and risk of pelvic organ prolapse. Int J Gynaecol Obstet 2008;103:55-8.
Bonin S, Hlubek F, Benhattar J, et al. Multicentre validation study of nucleic acids
extraction from FFPE tissues. Virchows Arch 2010;457:309-17.
Norton N, Williams NM, Williams HJ, et al. Universal, robust, highly quantitative
SNP allele frequency measurement in DNA pools. Hum Genet 2002;110:471-8.
Figure S1 Determination of the sensitivity of the collagen detection SNaPshot assay.
A: Both alleles (G-allele in blue colour and A-allele in green colour from exon 30) are
present, derived from a dilution of 99.9% G/G DNA (donor) and 0.1% G/A DNA (recipient).
In this setting, the primer concentration in the SNaPshot reaction was increased by 10 fold
(0.5 to 5 pmol), so a detection limit of 0.1% recipient DNA within the background of donor
DNA was reached. The arrow indicates the green peak derived by the recipient´s A-allele.
The pink bar indicates that the height of the fluorescence units of the blue peak is beyond the
linear range.
B: Representative data from analysis of collagen type III expression in donor heart of patient
4 using 10 fold primer concentration. Only a peak in blue colour, representing G-alleles from
donor-derived collagen was detectable with a detection limit of 0.1%
6
Table. Biopsies for which a collagen type III origin measurement were performed.
Pat.
1
2
3
7
Interval to
Acute rejection in
biopsy
current biopsy
(years)
(ISHLT)
1yr
1A
4yrs
1A
8yrs
1A (site of rebiopsy)
9yrs
1B
10yrs
1A
12yrs
0
13yrs
0 (site of rebiopsy)
14yrs
0
7 days
0
2 months
0
1yr
1A
4yrs
3B
8yrs
0 (site of rebiopsy)
9yrs
1A
10yrs
1B
11yrs
0 (site of rebiopsy)
3 days
1A
1 month
1A
1yr
1B
3yrs
1A
5yrs
0 (site of rebiopsy)
6yrs
1A (site of rebiopsy)
7yrs
1A
4
5
6
7
8
9yrs
0
3 days
2
1 month
1B
1yr
1A
4yrs
3A
7yrs
1B
10yrs
0 (site of rebiopsy)
11yrs
1A
12yrs
0 (site of rebiopsy)
1yr
1A
4yrs
1B
9yrs
0 (site of rebiopsy)
10yrs
1B
11yrs
1A
12yrs
1A (site of rebiopsy)
13yrs
1A
14yrs
0
1yr
1B
4yrs
3B
10yrs
0 (site of rebiopsy)
11yrs
1A
13yrs
0 (site of rebiospy)
14yrs
1A
14yrs
0
15yrs
0
Not
No differences in
analyzed
collagen genotype