Since January 2010, we have been conducting a prospective study (NCT03546062)  under ALCOA (Attributable, Legible, Contemporaneous, Original, and Accurate) integrity protocols with a follow-up of 12 months on patients who underwent their first HTX at the HTX referral center of Monaldi Hospital (Naples, Italy) following International Society for Heart and Lung Transplantation (ISHLT) guidelines . The Ethical Committee approved the study (protocol no. 438) and patients gave written informed consent. The study group consisted of 197 patients enlisted to undergo HTX and followed for 12 months (Fig. 1). All patients were treated with RAS-inhibitor drugs (ACE-I or ARB). The recipients’ patients, at baseline and follow-up, under ACE-I received either 5 mg, 10 mg or 20 mg of Lisinopril once daily and/or 5 mg, 10 mg or 20 mg of Enalapril once daily. The recipients’ patients, at baseline and at follow-up, under ARB received 50 mg, 100 mg, or 150 mg of Losartan once daily.
The study population was divided into two groups according to whether patients did or did not have T2DM before the transplantation. The study included patients with T2DM for at least 6 months before HTX, without diabetic complications, following ISHLT guidelines . Patients with endomyocardial biopsy specimens consistent with ISHLT Grade 2R are considered positive for rejection, donor-specific antibodies (DSA) and IgM and IgG cytomegalovirus antibodies and increased T4/T8 ratio as well as with post-HTX diabetes were excluded from the study. Details of the surgical technique employed and the pharmacological tools at the follow-up were previously reported .
Clinical and echocardiographic evaluations
The internationally accepted evaluations were recorded after HTX at weeks 1, 24, and 48 (clinical and instrumental evaluation and glycemic control, i.e., fasting glycemia and HbA1c). At 12-month follow-up, the patients were divided, as post hoc analysis, into non-T2DM, T2DM with good glycemic control (HbA1c < 7%), and T2DM in poor glycemic control (HbA1c ≥ 7%) groups, based on the mean HbA1c evaluated quarterly . Moreover, Ang 1–7 and Ang 1–9 levels in urine samples by ELISA, following the manufacturer’s protocol for biological fluids (MBS703599-96 and MBS2022456, MyBioSource), were determined. 24-h urine samples were collected at weeks 1 (Basal), 12 (Intermediate), and 48 (Final) in plastic containers by adding 20 ml of 6N HCl to completely inhibit the degradation of angiotensin peptides at room temperature for over 36 h . In addition, we performed echocardiographic evaluations of systolic [ejection fraction (EF) and tricuspid annular plane systolic excursion (TAPSE)] and diastolic (E/e′ ratio) heart function at baseline and after a 12-months follow-up, as previously described (Fig. 1) .
After HTX, all patients’ endomyocardial biopsies (EMBs) were obtained either as a routine surveillance protocol or as tools for diagnosing allograft dysfunction and clinically suspected rejection [15, 16]. The standard biopsy schedule was performed as follows: weekly for the first month, fortnightly for the next month, once in the next 4 weeks, once in the next 6 weeks, then every 3 months for the next 2 years, and after that, every 6 months (Fig. 1). Biopsies were performed as previously described . Endomyocardial biopsy specimens were analyzed for cellular viability by evaluating Hypoxia-inducible factor-1α (HIF-1α) without suspicion of histological rejection. Although the study was based on prospective biopsies of implanted hearts, an experienced thoracic surgeon excised four to six tissue specimens of about 5–10 mm3 from the left ventricular free wall. Tissues were immediately treated and analyzed as described previously .
The biopsy evaluations were performed at 1 week (Basal) and 48 weeks (Final) (Fig. 1).
Immunofluorescence detection of ACE2 was evaluated in deparaffinized explanted heart sections from non-T2DM, T2DM with good glycemic control (HbA1c < 7%), and T2DM in poor glycemic control (HbA1c > 7%). Briefly, antigen retrieval buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) was added to deparaffinized and rehydrated sections and boiled in the microwave for 20 min. Slides were washed in phosphate-buffered saline (PBS) followed by incubation for 30 min in Tris-buffered saline (TBS) containing 50 mM ammonium chloride to reduce background fluorescence. All sections were blocked for 1 h at room temperature (RT) in fetal bovine serum (FBS) with saponin (0.1 g/ml) and stained with primary antibodies against ACE2 (1:500, ab15348, Abcam) and Cardiac Troponin T [1C11] (1:500, ab8295, Abcam) for 16 h. Sections, incubated using Alexa Fluor 488 or 633 secondary antibodies diluted at 1:1000 in blocking solution for 1 h at RT, were then quenched for autofluorescence using the Vector TrueVIEW Autofluorescence Quenching Kit (VEC-SP-8500, Vector Laboratories). To ensure that what appears to be specific staining was not caused by non-specific interactions of immunoglobulin molecules with the sample, sections from non-T2DM and T2DM patients were incubated with blocking solution, supplemented with a non-immune immunoglobulin IgG antibody, followed by a secondary antibody incubation for 1 h at RT. All samples were stained with DAPI (4′,6-diamidino-2-phenylindole; 5 µg/ml) for 10 min before mounting in Vectashield Mounting Medium (Vector Laboratories, catalog no. H-1700). Using a Zeiss LSM 710 confocal microscope, all slides were imaged with a plan apochromat X63 (NA1.4) oil immersion objective.
The myocardial levels of GlycACE2 protein were evaluated in explanted heart samples and endomyocardial biopsies from non-T2DM and T2DM patients by immunoblotting analysis. As for the preparation of myocardial protein extracts, 2D lysis buffer (7 mol/l urea, 2 mol/l thiourea, 4% CHAPS [3-([3-cholamidopropyl] dimethylammonium)-1-propane sulfonate] buffer, 30 mmol/l Tris–HCl, pH 8.8), were added to tissues cut into small pieces. Tissues homogenized with a Precellys 24 system (Bertin Technologies) were centrifuged at 800×g for 10 min at 4 °C to collect the supernatant. 50–60 μg of sample proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Membranes were incubated for 1 h at RT with blocking buffer solution, TBS-T containing 20 mM Tris, pH 7.6, 100 nM NaCl, 0.1% Tween-20, and 5% non-fat dry milk under gentle shaker. Membranes were then incubated with specific primary antibodies against ACE2 (1:1000, ab15348, Abcam) or GlycACE2 (1:1000, #4355, Cell Signaling Technology) at 4 °C overnight, followed by incubation with peroxidase-conjugated secondary antibodies for 1 h at RT. In this study two antibodies have been used in order to distinguish GlycACE2 from ACE2. The antibody for ACE2 (ab15348, Abcam) detects a band size in human tissues at 120–135 kDa, as reported by the manufacturer. The antibody for GlycACE2 (#4355, Cell Signaling) detected a band at 120–135 kDa, and was also tested with an aliquot of recombinant human ACE2 (hACE2) (MW = 100 kDa) after in vitro glycation . As reported, hACE2 was separated on SDS-PAGE by using 7% gels in reducing and non-reducing conditions and then transferred to nitrocellulose membrane . Membrane incubated with antibody against GlycACE2 (1:1000) (#4355, Cell Signaling Technology) showed a band at a molecular weight higher than 100 kDa (about 135 kDa) supporting the non-enzymatic glycosylation of hACE2 protein. This evidence was strengthened by the detection of a band at 250 kDa under reducing conditions, corresponding to the dimer formation (Additional file 1: Fig. S1). Protein normalization was performed using α-tubulin (#2125, Cell Signaling, catalog no. 2125; 1:5000). The chemioluminescent reaction has been performed on a dried membrane to independently focus on non-glycosylated ACE2 or glycosylated ACE2 protein. Images were acquired by using Image Lab 5.2.1, Molecular Imager ChemiDoc XRS Imaging system (Bio-Rad Laboratories), and band densities were measured by ImageJ software (National Institutes of Health, Bethesda, USA) and expressed as arbitrary units (AU). The GlycACE2 content was evaluated as the percentage of the total amount of ACE2.
Real time-polymerase chain-reaction
Total RNA was isolated from human heart sample homogenates, according to the manufacturer’s protocol, by using RNeasy Mini kit (74106, Qiagen) and was quantified with NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific). Genomic DNA (gDNA) contaminations were removed from heart samples and mRNA was converted to cDNA by using QuantiTect Reverse Transcription kit (205311, Qiagen)—Reverse Transcription with Elimination of Genomic DNA for Quantitative, Real-Time PCR Protocol—and Gene AMP PCR System 9700 (Applied Biosystems). cDNA were amplified with the CFX96 Real-time System C1000 Touch Thermal Cycler (BIORAD), according to the protocol “Two-Step RT-PCR (Standard Protocol)”. Particularly, QuantiTect SYBR Green PCR Kit (204143, Qiagen) and QuantiTect Primer Assays were used in order to detect human ACE2 (ACE2—QT00034055, Qiagen) gene expression, quantized with 2−ΔΔCt method by using GAPDH (QT00079247, Qiagen) as control.
Ang 1–7, Ang 1–9, MasR, and NFAT
Enzyme-linked immunosorbent assay (ELISA) colorimetric kits were used for the determination of human Ang 1–7, Ang 1–9, MasR and NFAT (Human Ang 1–7 ELISA Kit, E-EL-H5518, Elabscience; Human Ang 1–9 ELISA Kit, EKU10061, Biomatik; Human MAS1 ELISA Kit, abx555483, Abbexa; Human NFAT activation molecule 1 (NFAM1) ELISA Kit, abx520337, Abbexa) levels in tissue extracts from heart biopsies (1 mg/ml of total protein), according to the manufacturer’s protocol for tissue homogenates. Briefly, tissues were rinsed in ice-cold PBS, cut into small pieces, and homogenized in fresh 2D lysis buffer with a Precellys 24 system (Bertin Technologies). The resulting suspension was centrifuged for 5 min at 10,000×g and the clarified surnatant was incubated in the pre-coated plates with specific anti-Ang 1–7, -Ang 1–9, -MAS1 and -NFAM1 antibodies, following the manufacturer's instruction. For each sample, the Optical Density (OD) is measured spectrophotometrically at 450 nm in a microplate reader (Bio-Rad) and Ang 1–7, Ang 1–9, MAS1 and NFAM1 levels in samples determined by plotting the absorbance values against concentrations of each standard curve. The assessment of Ang 1–7 and Ang 1–9 content was performed by using ELISA kits with high specificity in the detection to avoid significant cross-reactivity or interference between Ang 1–7 or Ang 1–9 and their analogs as reported in the specific datasheet. In detail, during the reaction, human Ang 1–7 or Ang 1–9 in samples compete with a fixed amount of human Ang 1–7 or Ang 1–9 on the solid phase supporter for sites on the biotinylated detection Ab specific to Human Ang 1–7 or Ang 1–9. No significant cross-reactivity or interference between human Ang 1–7 and Ang 1–9 was observed.
For morphological diagnosis, sections (4 μm thick) were stained with hematoxylin and eosin (H&E). Masson's Tricromica Stain was used for the differential staining of collagen. All stained samples were examined under light and digital microscopes. The content of collagen fibers relative to the total adjacent normal tissue by image analysis using the software Zen 3.3 (blue edition, Zeiss) was also evaluated.
Data are expressed as mean ± SD for continuous variables and percentage for categorical variables.Two-way repeated-measures ANOVA was conducted to determine the differences in cardiac GlycACE2, Ang 1–7, Ang 1–9, MasR, and NFAT levels at baseline and after 12 months in diabetic and non-diabetic patients. Interaction effect was assessed to determine within-group changes and between-group differences at baseline and 12 months. The Shapiro–Wilk test was used to assess the normality of the data. A multiple regression model was used to assess changes in GlycACE2 levels by age, sex, BMI, and glycated hemoglobin levels. A P-value < 0.05 was considered statistically significant. Data were analyzed with SPSS software (version 23).