Patients Cohort and Samples
Human myocardial samples were obtained from Type 2 diabetes mellitus (T2DM) and NON-Diabetic Mellitus (NDM) patients with post-infarct cardiomyopathy which undergoing surgical coronary revascularization. The samples were collected only when removal of tissue was required for surgical reasons. Collection of human tissues samples was approved by the local ethics committee at the University of Campania "L. Vanvitelli" of Naples. Before cardiac surgery, written informed consent was obtained from every patient. All patient data were kept anonymous. We included 10 T2DM and 6 NDM patients from which peri-infarct/border zone biopsies were obtained (Table 1). Freshly excised samples were formalin fixed for immunohistochemistry analysis as described below. Furthermore, additional 6 T2DM and 6 NDM patients with similar characteristics were included (Table 1), from which atrial samples were obtained and processed for cell harvesting as below described.
Human CSCs Isolation
Human samples obtained from T2DM and NDM patients were stored in saline on ice until ready to process (~1hr). All steps were performed at 4°C unless stated otherwise. Briefly, cardiac tissue was minced then digested with collagenase II (0.3mg/ml; Worthington Laboratories) in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich) at 37°C in a series of sequential digestions for 3 minutes each. Enzymatically released cells were filtered through a 40µm cell strainer (Becton Dickinson, BD) to eliminate the cardiomyocyte population and collected in enzyme quenching media (DMEM + 10% FBS). The isolated cardiac cells were collected by centrifugation at 400g for 10 min, resuspended in incubation media (PBS, 0.5% BSA, 2 mM EDTA). For the isolation of c-kitposCD45negCD31neg CSCs the MACS technology was used (Miltenyi Biotec). First, cardiomyocyte-depleted cardiac cells were negatively sorted for CD45pos and CD31pos cells by immunolabelling with anti-human CD45 and CD31 magnetic immunobeads (Miltenyi). The obtained CD45negCD31neg population was then enriched for c-kitpos cardiac cells through incubation with anti-human CD117 immunobeads (Miltenyi) (1:10) and then positively sorted using MACS according to the manufacturer’s instructions.
Cell Culture
Freshly isolated human c-kitposCD45negCD31neg CSCs were plated in CELL-Start (Life Technologies) coated dishes in complete CSC growth medium consisting of a 1:1 ratio of DMEM-F12-Ham’s Gibco, Life Technologies) medium and Neurobasal medium (Gibco, Life Technologies), implemented with Embryonic stem-cell FBS (10%, Gibco, Life Technologies), insulin-transferrin-selenium (1%, Life Technologies), epidermal growth factor (20 ng/ml, Peprotech), basal fibroblast growth factor (10 ng/ml, Peprotech), human leukemia inhibitory factor (10 ng/ml, Miltenyi), 37 mg of l-glutamine, B27 supplement (2%, Life Technologies), N2 supplement (1%, Life Technologies), penicillin-streptomycin (1%, Life Technologies), Fungizone (0.1%, Life Technologies) and gentamicin (0.1%, Life Technologies). Cells were maintained in a humidified hypoxic incubator at 37°C, 5% CO2, 2% O2. Media were replenished every 48 hours and cells were passaged at a 1:4 ratio.
Histology and Immunohistochemistry
Tissue specimens were fixed and embedded in paraffin for histochemical and immunohistochemical analysis. Human tissues were cut in sections of 5 µm. After dewaxing in xylene and rehydration in graded concentration of ethanol, when appropriate, Antigen Retrieval was performed by incubating section in 10 mM sodium citrate buffer (pH 6.0) at 98°C for 30 min. Non-specific antibody binding was blocked by incubation with 10% normal donkey serum (Jackson ImmunoResearch) for 30 minutes at room temperature. Sections were stained for 1h at 37°C or overnight at 4°C and the following primary antibodies were used: c-kit (1:100 dilution, DAKO), c-TnI (1:200 dilution, Abcam), p16INK4A (1:100 dilution, Santa Cruz Biotechnology), 8-OH-dG (1:100 dilution, Origene) and 3-NT (1:400 dilution, Merck Millipore). After washing in phosphate-buffered saline (PBS), sections were incubated with respective secondary antibodies (Jackson ImmunoResearch). Nuclear counterstains were performed by DAPI (4',6-diamidino-2-phenylindole, Sigma) and sections were examined by confocal microscopy (LEICA TCS SP8). The number of positive cells was expressed as a percent fraction of the total cells number per mm2.
For immunohistochemical analysis of oxidative stress on tissue sections, after dewaxing, human sections were blocked with Dual Endogenous Enzyme Block for 10 min at room temperature. Then, the sections were stained with antibodies against 8-OH-dG (1:100 dilution, Origene), 3-NT (1:400 dilution, Merck Millipore) and 4-HNE antibody (1:50 dilution, Abcam) for 1h at 37°C. Positive reactions were visualized using a Labelled Polymer-HRP complex and 3,3’-diaminobenzidine tetrahydrochloride (DAB) chromogen using the EnVision+ Dual Link System-HRP (DAKO). Sections were then counterstained with hematoxylin, permanently mounted, and then examined with light microscopy (LEICA, DMI3000B). The morphometric analysis of immunohistochemistry was conducted using Image J.
FACS analysis
Cell analysis was performed on FACSCanto II (BD) with FlowJo software (TREE STAR) to identify the percentage of cardiac small cells expressing different cell-surface markers of interest at passage 2 (P2). A panel of different markers was used for immunophenotypic characterization of human c-kitposCD45negCD31neg CSCs obtained from T2DM and NDM patients. Specific antibodies used are listed in Table 2. Appropriate labelled isotype controls were used to define the specific gates.
Proliferation, clonogenicity, cardiosphere formation and cardiomyocyte differentiation assays in vitro
CSC proliferation was evaluated through BrdU incorporation and growth curve assay, at the indicated time points, on human CSCs. To assess the proliferative activity of freshly isolated c-kitposCD45negCD31neg T2DM-hCSCs and NDM-hCSCs, BrdU 10 μM was administered in vitro. CSCs were plated at density of 1x103 in 24-well CELL-Start-coated dishes and then serum starved in 0% serum base medium. After 48 hrs, starvation medium was replaced by CSC growth medium and BrdU was added to the medium every 6hrs. The cells were fixed at 24 hours and BrdU incorporation was assessed using the BrdU detection system kit (Roche) according to the manufacturer’s instructions. Nuclei were counterstained with DAPI (Sigma-Aldrich). Cells were evaluated using a fluorescent microscope (LEICA, DMI3000B). Accordingly, the number of BrdUpos cells was expressed as a percent fraction of the total cell nuclei.
Growth curve assay was archived by plating 5×103 cells in 24-well CELL-Start-coated dishes in CSC growth medium and then serum starved in 0% serum base medium. After 48 hrs, starvation medium was replaced by CSC growth medium and cells were then trypsinized and counted using trypan blue, 1:1 ratio, at the indicated time points.
Single cell cloning was employed through depositing of half c-kitposCD45negCD31neg T2DM-hCSCs and NDM-hCSCs into 96-well CELL-Start-coated Terasaki plates by serial dilution. Individual c-kitposCD45negCD31neg T2DM-hCSCs and NDM-hCSCs were grown in CSC growth medium for 1–3 weeks when clones were identified and expanded. The clonogenicity of the c-kitposCD45negCD31neg T2DM-hCSCs and NDM-hCSCs was determined by counting the number of wells in each 96-well plate containing clones and expressed as a percentage. A total of 10 plates were analyzed.
For cardiosphere generation, 1x105 c-kitposCD45negCD31neg T2DM-hCSCs and NDM-hCSCs were placed in bacteriological dishes with CSC growth medium. Cardiospheres were counted per plate at 14 days and the number expressed as a percentage relative to the number of plated CSCs.
For specific myogenic differentiation, c-kitposCD45negCD31neg T2DM-hCSCs and NDM-hCSCs-derived-cardiospheres were switched to StemPro®-34 SFM differentiation medium (a serum-free medium conditioned with StemPro®-Nutrient Supplement, Gibco, Life Technologies), Glutamine (2mM) and penicillin-streptomycin (1%, Life Technologies). For cardiomyocyte differentiation BMP4 (10ng/ml, Peprotech), Activin-A (50 ng/ml, Peprotech), β-FGF (10ng/ml, Peprotech), Wnt-11 (150ng/ml, R&D System) and Wnt-5a (150ng/ml, R&D System) were added to base differentiation medium. Then differentiating cardiospheres were pelleted and transferred to laminin coated dishes (1µg/ml) and Dkk-1 (150ng/ml, R&D System) was added to base differentiation medium until day 14. Cell differentiation was evaluated at 14 days.
Immunocytochemistry
A volume of 200 μl of a 0.15×106 cells/ml suspension of c-kitposCD45negCD31neg CSCs obtained from T2DM and NDM patients were directly loaded in cytofunnel and spin down at 800 rpm for 3 min onto poly-lysine-coated slides using a Shandon Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific). Slides were immediately fixed using PFA 4% (Sigma-Aldrich). After fixation, cells were allowed to air dry before proceeding with immunostaining. Slides were washed with PBS, and incubated in 0.1% Triton X-100:PBS at room temperature for 10 minutes. After washing with 0.1% tween:PBS and 30 minutes in 10% donkey serum, cells were incubated overnight at 4°C with primary antibodies against p16INK4a (1:100 dilution, Santa Cruz Biotechnology), γ-H2AX (1:400 dilution, Cell Signaling) in 0.1% Tween:PBS. Slides were then washed and incubated with corresponding secondary antibodies (Jackson Immunoresearch) for 1 hour at 37°C. After washing, the nuclear DNA of the cells was counterstained with DAPI (Sigma-Aldrich) at 1µg/ml and mounted using Vectashield mounting media (Vector labs). Imagines were acquired using a confocal microscope (LEICA TCS SP8). The number of p16INK4a positive and γ-H2AX positive cells was expressed as a percent fraction of the total CSC nuclei. Cell apoptosis were detected using the Terminal deoxynucleotidyl Transferase (TdT) assay (TUNEL assay, Merck Millipore) according to the manufacturer’s instructions. Briefly, to perform TUNEL staining, cells were fixed with paraformaldehyde 2% in PBS (pH 7.4) and permeabilized with 0.1% Triton X–100 in 0.1% sodium citrate. Then cells were incubated with TUNEL reaction mixture (containing Terminal deoxynucleotidyl transferase enzyme + nucleotide mixture in reaction buffer) for 1 hour at 37°C in a humidified atmosphere in the dark. After washing, the nuclear DNA of the cells was counterstained with DAPI (Sigma-Aldrich) and the slides mounted using mounting media (Vector labs). Imagines were acquired using a confocal microscope (LEICA TCS SP8). The number of apoptotic cells was expressed as percentage of the total nuclei.
SA–β-gal staining was performed using a SA–β-gal staining kit (Cell Signaling Technology) according to the manufacturer's instructions and protocols previously reported [23]. In brief, c-kitposCD45negCD31neg T2DM-hCSCs and NDM-hCSCs were grown in culture and then fixed with 1X Fixative Solution for 10-15 min at room temperature. Following fixation, cells were washed with PBS before being incubated in β-Galactosidase Staining Solution (pH 6.0) at 37°C in a dry incubator overnight. The enzymatic reaction was stopped by washing slides with ice-cold PBS and SA–β-gal staining was fixed with ice-cold methanol for 30s before mounting/visualising. Senescent CSCs were identified as blue-stained cells using light microscopy. A minimum of 10 images were taken at x10 magnification from random fields and the percentage of SA–β-gal cells were expressed as percentage of total nuclei.
To perform cTnI staining, human c-kitposCD45negCD31neg CSCs-derived cardiospheres were fixed using PFA 4% (Sigma-Aldrich) after 14 days in differentiation media. After fixation, cells were incubated with cTnI antibody (1:200, Abcam) for 2 hour at 37°C in a humidified atmosphere. The corresponding secondary antibody (Jackson Immunoresearch) was added for 1 hour at 37°C in the dark. The nuclear DNA of the cells was counterstained with DAPI (Sigma-Aldrich) and the slides mounted using mounting media (Vector labs). Imagines were acquired using a confocal microscope (LEICA TCS SP8).
Telomere length
Genomic DNA of human c-kitposCD45negCD31neg CSCs obtained from myocardial samples of T2DM and NDM patients was extracted using Quick-DNA Microprep Kit (Zymo Research). Telomere length was analyzed by using the Absolute Human Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories). Genomic DNA (10 ng) was amplified with the FastStart DNA Green Master (Roche Life Science) using a CFX384-Real-Time PCR System (Biorad) and data analysis was conducted according to manufacturer’s instruction. For each genomic DNA samples, two different reaction were performed using two primer set: a Telomere primer set to recognize and amplify the telomere sequences and a single copy reference (SCR) primer set to normalize the data that recognizes and amplifies a 100 bp-long region on human chromosome 17, accordingly, a known telomere length as reference to calculate the telomere length of target samples. All reactions were run in triplicate. The average telomere length was calculated by following the manufacturer’s instructions.
Telomerase Activity Quantification
Human c-kitposCD45negCD31neg CSCs obtained from myocardial samples of T2DM and NDM patients were processed according to the manufacturer’s protocol using the Telomerase Activity Quantification qPCR Assay Kit (ScienCell Research Laboratories) [24]. Briefly, cell pellets were thawed in lysis reagent enables to release telomerases in the native state. Cell lysate samples were incubated with telomerase reaction buffer at 37°C for 3 hours. qRT-PCR was conducted with FastStart DNA Green Master (Roche Life Science) using a CFX384-Real-Time PCR System (Biorad) and data analysis was performed according to manufacturer’s instruction. The telomere primers set recognizes and amplifies newly synthesized telomere sequences in the assay. All reactions were run in triplicate. Telomerase activity quantification was calculated by following the manufacturer’s instructions.
RNA extraction and RT-PCR analysis
RNA was extracted from human c-kitposCD45negCD31neg CSCs obtained from myocardial samples of T2DM and NDM patients, using TRIzol® RNA Isolation Reagents (Ambion) and quantitated using a Nanodrop 2000 Spectrophotometer (Thermo Fisher scientific). Reverse transcription was performed with 0.5-1 µg of RNA using HighCapacity cDNA Kit (Applied Biosystem). Quantitative RT-PCR was performed using TaqMan Primer/Probe sets (see Table 3) using StepOne Plus real Time PCR System (Applied Biosystems). The following genes were tested: GATA-4, NKX2.5, MEF2C TNNT2, ACTC1, MYH6, MYH7, MCP1, IGFBP5, MMP3, IL-6, CCL11, IL-8, GM-CSF and PAI1. Data were processed by the ΔCt method using StepOne Software v2.3 and mRNA was normalized to the housekeeping gene, GAPDH. All reactions were carried out in triplicate.
Western Blot Analysis
Immunoblots were carried out using protein lysates obtained from c-kitposCD45negCD31neg T2DM-hCSCs and NDM-hCSCs. Aliquot equivalent of ~40 to 70 μg of proteins were separated on gradient (6–15%) SDS-polyacrylamide gels. After electrophoresis, proteins were transferred onto nitrocellulose filters, blocked with either 5% dry milk and incubated with Ab against p16INK4A (1:1000, Santa Cruz Biotechnology) and GAPDH (1:1000, Santa Cruz Biotechnology). Proteins were detected by chemiluminescence using horseradish peroxidase-conjugated 2Abs and placing the nitrocellulose filters on a photographic film. The acquisition was archived using Medical X-ray processor 2000 (CARESTREAM). Densitometry was obtained using ImageJ software. Immunoblots were performed in biological triplicates.
Cytokine & growth factors assay
Cytokine and growth factor levels [Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interferon γ (IFNγ), tumor necrosis factor alpha (TNFα), Monocyte Chemoattractant Protein-1(MCP-1), Vascular Endothelial Growth Factor (VEGF), Epidermal growth factor (EGF)] were simultaneously evaluated through the “Cytokine & Growth Factors Array (CTK)” kit (Randox Labs) by using Evidence Investigator biochip analyzer (Randox Labs) [25], a chemiluminescent immunoassay in which antibodies bonded on the biochip surface allow the immobilization of a specific analyte, subsequently bonded with a secondary antibody labelled with horseradish peroxidase (HRP), to trigger a luminol-based chemiluminescent signal emission. The light signal generated from each of the test regions is then detected using imaging technology and quantified with a stored calibration curve.
Senolytic drug treatment, viability in vitro
c-kitposCD45negCD31neg T2DM-hCSCS were plated in 24-well CELL-Start-coated plates at 40% confluence and left for 72h. Then, the growing medium was replaced with new fresh medium in the absence or presence of a combination of two senolytics drug, Dasatinib (D, LC Laboratories) and Quercetin (Q, Sigma-Aldrich), respectively at doses 0.25μM and 10μM added for 6 hours. Then, the conditioned medium was replaced with complete fresh medium. After 48h the treatment D+Q was repeated as above.
For myogenic differentiation, T2DM-hCSCs were treated or untreated with D+Q in growth media as above. Then cells were placed in bacteriological dishes and the differentiation assay was performed as above described.
Statistical analysis
All data are presented in mean ± standard deviation. Data were analysed using t-test comparisons in GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com. Differences of p<0,05 are considered statistically significant.