Human cardiac biopsies
The study was approved by the Research Ethics Board at the Sahlgrenska Academy, University of Gothenburg, Sweden, following the Helsinki Declaration. This study was based on whole explanted hearts from which biopsies were obtained from the anterolateral wall of the left ventricle (LV).
Two groups of research subjects were included through collaboration with the Transplant Institute at Sahlgrenska University Hospital. In the first group, cardiac tissue from 13 multi-organ donors was obtained. The hearts were not suitable for heart transplantation but explanted for homograft procurement and used in the present study after the valves were harvested. Organ donors with chronic heart failure were excluded. All had documentation of consent from the donor, stating that their organs could be used for other medical purposes than organ donation. The second group included 11 patients with severe heart failure undergoing cardiac transplantation. After signed informed consent was obtained, cardiac tissue was harvested from the hearts that were removed during cardiac transplantation surgery. The clinical background is summarized in Tables 1 and 2.
Relative protein quantification
Tissue from six organ donors (age 19–63 years) and from four patients (age 28–65 years) in the heart failure group was used. The tissue was snap-frozen and stored at −80 °C until use.
Proteins were extracted using a lysis buffer (50 mM triethylammonium bicarbonate (TEAB), 2% sodium dodecyl sulfate (SDS)). 30 μg from each sample and a reference sample were digested into peptides using filter-aided sample preparation (FASP)16. The reference consisted of aliquots from each group to provide representative references. Peptides were labeled using TMT 11-plex isobaric mass tagging reagents (Thermo Fisher Scientific), according to the manufacturer’s instructions. The TMT set was fractionated using basic reverse phase liquid chromatography (pH 10) into 20 fractions and analyzed in an Orbitrap Fusion Tribrid mass spectrometer interfaced with the Easy-nLC1200 liquid chromatography system (Thermo Fisher Scientific). The peptides were separated on an analytical C18 column using a gradient from 4 to 28% acetonitrile in 0.2% formic acid over 75 min. Relative quantification was performed using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) and the Mascot search engine (v. 2.5.1 Matrix Science, London, UK) matching against the SwissProt H. sapiens database (July 2019). Peptide and fragment tolerances were set to 5 ppm and 0.6 Da, zero missed cleavages, variable methionine oxidation, fixed cysteine methylthiolation and TMT-6 modifications on lysine and peptide N-termini. Percolator was used for PSM validation at the 1% FDR threshold. TMT reporter ions were identified in the MS3 HCD spectra with a 3 mmu mass tolerance, and samples were normalized on the total peptide amount. The reference sample was the denominator and used to calculate abundance ratios. Statistical analysis was performed with Perseus software17 (version 1.6.10.45), two-side students t-test on the Log2 protein abundance ratios for p value calculations. For the volcano plot the number of randomization was set to 250, FDR to 0.05 and S0 to 0.1.
RNA isolation and gene expression by qPCR
Left ventricular tissue was obtained from 14 organ donors (age 19–74 years) and 11 patients with severe heart failure in conjunction with heart transplantation (age 27–67 years). The tissue was preserved in RNAlater. Total RNA was extracted using the reagents and equipment from Qiagen. Briefly, the tissue was homogenized with TissueLyser LT and Qiazol and then purified using a RNeasy Mini column with DNase1 treatment for removal of residual genomic DNA. The cDNA was prepared from total RNA using a High-Capacity cDNA reverse transcription kit with RNase Inhibitor #4374967 and TaqMan Gene Expression Master Mix #4369542 (Applied Biosystems).
The human TaqMan gene expression assay ACE2 Hs01085333_m1 was used for the gene of interest, and PPIA Hs99999904_m1 as the reference gene. The relative comparative method was used to analyze the RT-qPCR data (Sequence Detector User Bulletin 2, Applied Biosystems) and the relative quantification (RQ) values were calculated using PPIA as the reference gene and an in-house calibrator sample. Gene expression data are presented in relative units.
Immunohistochemistry
Biopsies from the LV from seven organ donors and six heart failure patients were embedded in Tragacant mounting medium (Histolab Products AB, Gothenburg, Sweden), frozen in liquid nitrogen and stored at −80 °C. The frozen tissues were sectioned into 7 μm serial sections that were fixed in −20 °C acetone for 10 min and washed in Phosphate Buffer Saline (PBS). A 30-min blocking step followed, with 2% bovine serum albumin, 0.3% Triton–X100 and 5% goat serum (Invitrogen, Carlsbad, CA, USA) diluted in PBS.
Primary antibodies were diluted according to Table 3, added to the sections and incubated at 4 °C overnight in a humidified chamber. Results were visualized by staining with secondary antibodies: goat anti-rabbit Alexa Fluor 546 or goat anti-mouse Alexa Fluor 647 (Invitrogen) for 1–2 h at RT. To enable triple staining, cTnT antibody was conjugated with Alexa 488 using the Zenon kit (Invitrogen) and a 1:6 molar ratio. After incubation with secondary antibodies, the samples were washed and incubated with the Zenon conjugated cTnT antibody for another hour. Sections were fixed with Histofix (Histolab) for 15 min and mounted with Prolong Gold Antifade reagent with DAPI (Invitrogen). Corresponding isotype controls for the primary antibodies were used for determining the background and did not show any specific staining.
Image analysis and quantification
The results were visualized using an ECLIPSE Ti inverted microscope (Nikon Corporation, Tokyo, Japan). A Nikon DS-2Mv camera was used for brightfield histology images. For analyses of immunohistochemistry, fluorescence images were acquired with an Andor Zyla camera. Large images: 7 × 7 fields shot with the 20× objective were scanned at three Z levels to capture all parts of the large images in focus. Generally, four channels (DAPI, Alexa 488, Alexa 546 and Alexa 647) were acquired.
All images were exported to Image J software (v. 1.47 h, Fiji distribution)18 for further analysis. For each channel, displayed pixel ranges were set so that most of the background was extinguished. Isotypic controls were treated in the same way. The composite photos were used for analysis of the expression of biomarkers. Two large images, composed of stitched photos of 49 fields by 20× objective were analyzed for each individual.
For quantification of ACE2 expression, an intensity threshold was first set for each staining using a customized plugin, to reduce background staining. Pixel values below the threshold were set to zero. The threshold was then subtracted from pixel values above the threshold in order to get a continuous distribution of pixel values. All images were treated equally. The threshold level was set based on background staining of isotype control images. Areas of quantification were created include most of the stained tissue, but excluding obvious artefacts and parts of the images not in focus (Suppl. Figure 1). Mean pixel intensity measurements were carried out on ACE2 expression for each image. Outliers were excluded based on the 1.5 interquartile range (IQR) method. In total, measurements of 3 images were regarded as outliers (two donors, one heart failure subject), and were excluded. In each case, the other replicate image was not regarded as outlier. After outlier exclusion, a mean pixel intensity was calculated for each study subject. Difference in ACE2 expression between donors and heart failure subjects were tested using Wilcoxon rank sum test. P < 0.05 was considered significant. All statistical calculations were carried out using R v. 4.0.2 (R Core Team 2020 https://www.R-project.org/).
