WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2

Chemical

WIN 55,212-2 mesylate was purchased from TargetMol (T4458). Stock solutions were prepared using 100% dimethyl sulfoxide (DMSO; D2650-Sigma-Aldrich, St. Louis, MO, USA) and sterile-filtered. The final concentration of DMSO in work solution was 0.01%.

iPS-cardiomyocyte differentiation and purification

hiPSC-CMs were purchased from Pluricell (São Paulo, Brazil) and used between day 25 and day 35 of differentiation. The hiPSC-CMs used here were generated and previously characterized in vitro by Cruvinel et al. (2020). Briefly, the enrichment of the cardiomyocyte population was assessed by flow cytometry and immunofluorescence of TNNT2, a specific marker, which revealed that, on average, 88.4% (+/− 8.4%) of cells were positive cells (Fig. S4). hiPSC-CMs were handled in four different groups: MOCK and SARS-CoV-2 (SARS-CoV-2 infection without WIN), which were also analyzed as controls in Salerno et al. (2021), MOCK WIN (no SARS-CoV-2 infection + WIN), and SARS-CoV-2 WIN (SARS-Cov-2 infection + WIN). All WIN-treated hiPSC-CMs were pretreated for 24 h with one µM WIN. Fresh culture medium with (or without) one µM WIN, combined or not with SARS-CoV-2, was added for 24 h to each experimental group, respectively.

SARS-CoV-2 propagation

SARS-CoV-2 was expanded in Vero E6 cells from an isolate of a nasopharyngeal swab obtained from a confirmed case in Rio de Janeiro, Brazil (GenBank accession no. MT710714). Viral isolation was performed after a single passage in 150 cm² flasks cultured with high glucose DMEM plus 2% FBS. Observations for cytopathic effects were performed daily and peaked 4 to 5 days after infection. All procedures related to virus culture were handled in biosafety level 3 (BSL3) multi-user facilities according to WHO guidelines. Virus titers were determined as plaque-forming units (PFU/mL) as explained below, and virus stocks were kept at −80 °C.

SARS-CoV-2 titration

For virus titration, monolayers of Vero E6 cells (2 × 10⁴ cell/well) in 96-well plates were infected with serial dilutions of supernatants containing SARS-CoV-2 for 1 h at 37 °C. A semi-solid high glucose DMEM medium containing 2% FSB and 2.4% carboxymethylcellulose was added and cultures were incubated for 3 days at 37 °C. Then, the cells were fixed with 10% formalin for 2 h at room temperature. The cell monolayer was stained with 0.04% solution of crystal violet in 20% ethanol for 1 h. Plaque numbers were scored in at least three replicates per dilution by independent readers blinded to the experimental group and the virus titers were determined by plaque-forming units (PFU) per milliliter.

SARS-CoV-2 infection

hiPSC-CMs were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 in high glucose DMEM without serum. After 1 h, cells were washed and incubated with Complete Medium with or without treatments for 48–72 h. Next, the supernatant was collected and cells were fixed with 4% paraformaldehyde (PFA) solution for posterior analysis.

Measurement of cytokines mediators and LDH cytotoxicity

Cytokines (IL-6, IL-7, IL-8, and TNF-α) were quantified in the supernatants from hiPSC-CMs samples by ELISA (R&D Systems, Minneapolis, MN, USA) following manufacturer’s instructions. The analysis of data was performed using software provided by the manufacturer (Bio-Rad Laboratories, Hercules, CA, USA). A range of 0.51–8,000 pg/mL recombinant cytokines was used to establish standard curves and the sensitivity of the assay. Cell death was determined according to the activity of lactate dehydrogenase (LDH) in the culture supernatants using a CytoTox® Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

Gene expression analysis

Qualitative endpoint PCR reactions were executed with the following primer sequences: CB1 (forward 5′-ATGTGGACCATAGCCATTGTG-3′; reverse: 5′-CCGATCCAGAACATCAGGTAGG-3′) and CB2 (forward 5′-GCTATCCACCTTCCTACAAAGC-3′; reverse: 5′-CTCAGCAGGTAGTCATTGGGG-3′). Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH; forward: 5′-TTCGACAGTCAGCCGCATC-3′; reverse: 5′-GACTCCACGACGTACTCAGC-3′) was used as the endogenous housekeeping control gene. Each PCR reaction was performed in a ten μL mixture containing 0.25 U GoTaq G2 Hot Start Polymerase (Promega, Madison, WI, USA), 1 × GoTaq G2 Buffer, 1.5 mM MgCl2 (Invitrogen, Waltham, MA, USA), 200 nM of each primer (forward and reverse), 200 μM dNTP mixture containing the four deoxyribonucleotides (dATP, dCTP, dTTP, and dGTP), and ten ng of cDNA template. Appropriate negative controls and genomic DNA positive controls were incorporated into each experiment. Amplification thermal program included an initial denaturation step of 95 °C for 3 min and 40 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 15 s using the ProFlexTM PCR System Thermal Cycler (Applied Biosystems, Waltham, MA, USA). Subsequently, the total amount of PCR product was separated by electrophoresis at 110 V for 40 min in 1.8% agarose gel diluted in 1 × Tris-acetate EDTA buffer (w/v) and stained with 0.01% of SYBR Safe (Thermo Fisher, Waltham, MA, USA).

For real-time quantitative PCR, the reactions were carried out in triplicates in a reaction mixture containing 1 × GoTaq qPCR MasterMix (Promega Corporation, Madison, WI, USA), 300 nM CXR Reference Dye, a final concentration of 200 nM of each (forward and reverse) SYBR green-designed primers (Thermo Fisher Scientific, Waltham, MA, USA), and ten ng of cDNA template per reaction. Appropriate negative controls were added in each run. The relative expression of the genes of interest: ACE2 (forward: 5′-CGAAGCCGAAGACCTGTTCTA-3′; reverse: 5′-GGGCAAGTGTGGACTGTTCC-3′), MYH6 (forward: 5′-GCCCTTTGACATTCGCACTG-3′; reverse: 5′-GGTTTCAGCAATGACCTTGCC-3′), MYH7 (forward: 5′-TCACCAACAACCCCTACGATT-3′; reverse: 5′-CTCCTCAGCGTCATCAATGGA-3′) was normalized by human reference genes: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH; forward: 5′-GCCCTCAACGACCACTTTG-3′; reverse: 5′-CCACCACCCTGTTGCTGTAG-3′) and Hypoxanthine Phosphoribosyltransferase 1 (HPRT-1; forward 5′-CGTCGTGATTAGTGATGATGAACC-3′; reverse: 5′-AGAGGGCTACAATGTGATGGC-3′). The reactions were performed on a StepOnePlusTM Real-Time PCR System thermocycler (Applied Biosystems, Waltham, MA, USA). Thermal cycling program comprised of a denaturing step at 95 °C for 3 min, followed by 40 cycling stages at 95 °C for 15 s, 57 °C for 15 s, 72 °C for 15 s and melt curve stage 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s. Data analysis was performed with LinRegPCR quantitative PCR data analysis program v. 2020.0, as previously described.

Immunofluorescence staining

SARS-CoV-2-infected and mock-treated hiPSC-CMs were fixed using 4% paraformaldehyde solution (Sigma-Aldrich, EUA, St. Louis, Missouri, USA) for 1 h and stored at 4 °C. Next, cells were washed with PBS and then incubated with permeabilization/blocking solution (0.3% Triton X-100 /1% bovine serum albumin + 3% normal goat serum) for 1 h. Cardiomyocytes were incubated with primary antibodies diluted in a blocking buffer solution at 4° overnight: anti-SARS-CoV-2 convalescent serum from a positive COVID-19 patient (1:1,000) and anti-cardiac troponin T (TNNT2) (1:500, MA5-12960; Invitrogen, St. Louis, Missouri, USA). Afterwards, cardiomyocytes were incubated with the secondary antibody diluted in a blocking buffer solution: goat anti-Human Alexa Fluor 647 (1:400; A-21445; Invitrogen, St. Louis, Missouri, USA) and goat anti-Mouse 594 (1:400; A-11032; Invitrogen, St. Louis, Missouri, USA) for 1 h. Actin filaments were stained with Alexa Fluor 568 phalloidin (1:10; A-12380; Life Technologies, Carlsbad, California, USA) for 1 h. Nuclei were stained with 300 nM 4′-6-diamino-2-phenylindole (DAPI) for 5 min and each well was mounted with two drops of 50% PBS-Glycerol. Images (at least ten fields per well) of hiPSC-CMs were acquired using Operetta® High-Content Imaging System (Perkin Elmer) with a 20 × long working distance (WD) objective lens. A Leica TCS-SP8 confocal microscope was used to acquire images of hiPSC-CMs immunostained for TNNT2 and F-actin with the 63 × objective (Fig. S4).

Neutral red uptake cell viability assay

Briefly, hiPSC-CMs were seeded in 96-well plates. After reaching 80–90% confluency, cells were exposed to concentrations of WIN ranging between 10 nM–10 μM for 72 h. Next, the medium was replaced, cells were washed with PBS 1× and 200 μL of neutral red dye diluted in the hiPSC-CMs medium was added to each well at a final concentration of 0.05 mg/mL. After 3 h of incubation at 37 °C, neutral red dye was removed, and the cells were washed again. Then, 100 μL of the neutral red desorb solution was added (1% acetic acid-49% ethanol) to the wells, followed by 20 min in orbital shaking. Absorbance at 540 nm was measured with a Tecan Infinite^® 200 PRO (Life Sciences, Switzerland) spectrophotometer.

Western blotting

Twenty-four hours after treatment with WIN of hiPSC-CMs in 24-well plates, 100 µL of sample buffer without bromophenol blue (62.5 mM Tris-HCl, pH 6.8, containing 10% glycerol, 2% SDS, and 5% two-mercaptoethanol) was added in each well, and a cell scraper was used to help lyse the cells. Cell extracts were transferred to an Eppendorf tube, boiled at 95 °C for 5 min, and centrifuged at 4 °C 16,000 ×g for 15 min to collect the supernatant. Protein content was estimated using the Bio-Rad Protein Assay (# 5000006; Biorad, Hercules, CA, USA). Next, bromophenol blue (0.02%) was added, and extracted samples (40 µg/lane) were separated by an 8% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk in Tris Buffered Saline with 0.1% Tween-20 (TBS-T) for 1 h at room temperature. Then, membranes were incubated overnight at 4 °C with primary antibodies anti-ACE2 (1: 1,000; MA5-32307; Thermo Fisher, Waltham, MA, USA), anti-CB1 (1:300; CSB-PA007048, Cusabio), and anti-ACTIN (1:2,000; MAB1501; Millipore, Burlington, MA, USA), diluted in TBS-T with 5% non-fat milk. Membranes were washed and incubated with peroxidase-conjugated antibodies IgG (H + L), HRP-conjugate: goat anti-mouse (1: 10,000, G21040; Molecular Probes, Eugene, OR, USA), goat anti-rabbit (1: 10,000, G21234; Molecular Probes, Eugene, OR, USA), and rabbit anti-goat (1: 2,000, 61–1620; Invitrogen, Waltham, MA, USA) for 2 h at room temperature. The signals were developed using an ECL Prime Western Blotting System (# GERPN2232; Sigma, St. Louis, Missouri, USA) for 5 min, and chemiluminescence was detected with an Odyssey-FC Imaging System^® (LI-COR Biosciences, EUA). After CB1 or CB2 detection a stripping protocol was used on the membranes for further detection of actin. Membranes were incubated with a stripping buffer (pH 2.2, 200 mM glycine, 0.1% SDS, and 1% Tween-20) for three cycles of 10 min. Next, the buffer was discarded, and the membranes were washed three times with PBS and three times for 5 min with 0.1% TBS-T. Then, membranes were blocked again and proceeded with the above-described steps.

Statistics

Statistical analyses were performed using GraphPadPrism software version 8.0 (GraphPad, EUA). Results were expressed as the mean and standard error of the mean (SEM). For comparisons between two experimental groups, unpaired two-tailed Student’s t-test or Mann-Whitney U test was used, whereas two-way analysis of variance (ANOVA) or Kruskal–Wallis test followed by Tukey’s test was used for comparisons between three or more groups. A p-value smaller than 0.05 was accepted as statistically significant.

Ethics statement

Approved by the Research Ethics Committee of D’Or Institute of Research and Education (IDOR) 39474020.8.0000.5249.

Human cardiomyocytes express cannabinoid receptor 1 but WIN does not modulate ACE2 expression

As a first step to investigate the influence of cannabinoid receptors in SARS-CoV-2 infection of human cardiomyocytes, we checked whether hiPSC-CMs expressed CB1 and CB2 receptors. We found that hiPSC-CMs express only CB1 receptor mRNA (Fig. S1A), which was confirmed by CB1 protein expression (Fig. S1B). The banding pattern observed in the hiPSC-CMs was similar to the mouse hippocampus sample (positive control) and consistent with what was observed in samples from other CNS regions (Medina-Vera et al., 2020).

WIN is an agonist at the CB1 and CB2 receptors with a higher affinity to them than other cannabinoids (Acheson et al., 2011; Sachdev et al., 2019), including THC (Felder et al., 1995) and therefore it is an useful pharmacological tool to study cannabinoid receptor activation. Beforehand, we tested multiple WIN concentrations in readouts of cellular toxicity and permanent cardiac hypertrophy. We found that WIN did not reduce cell viability in concentrations up to one µM (Fig. S3A). Also, compared with control, one µM WIN did not increase MYH6 and MYH7 mRNA levels (Fig. S3B), genes that, when upregulated, may indicate cardiac hypertrophy in vitro (Wenzel, 1967; Rahmatollahi et al., 2016; Albakri, 2019). Therefore, we chose one µM WIN as the usage concentration for further assays since it caused neither cell death nor changes in gene expression related to hypertrophy.

After confirming that hiPSC-CMs express CB1 and ACE2 (Salerno et al., 2021), we asked whether WIN modulates ACE2 expression and, subsequently, influences SARS-CoV-2 infection within hiPSC-CMs. The cells were pretreated with one µM WIN for 24 h and analyzed for both mRNA and protein levels of ACE2. We observed that WIN-treated (1.15 ± 0.07 A.U.) and untreated (0.98 ± 0.11 A.U.) hiPSC-CMs had comparable levels of ACE2 mRNA whereas ACE2 protein levels in WIN treated cells were 1.16 ± 0.39, normalized to control (Figs. 1A, 1B and S2).

WIN does not influence SARS-CoV-2 infection and replication in hiPSC-CMs

Next, we asked whether WIN could reduce hiPSC-CMs SARS-CoV-2 infection by mechanisms other than ACE2 modulation. For this, cells were pretreated with one µM WIN for 24 h and infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 for 1 h, and the PFU analyzed 48 h later. In this study, we defined the use of MOI 0.1 for all experiments because this MOI had already been successfully used to infect hiPSC-CMs with SARS-CoV-2 (Sharma et al., 2020). Additionally, MOIs above 0.1 may not be a clinically plausible viral load found in vivo. Forty-eight hours after infection, we quantified convalescent serum (CS)-immunostaining and, as expected, we found that cells in the MOCK group had no CS immunoreactivity. Among the SARS-CoV-2-infected cells, those pretreated with WIN had a comparable percentage of infected cells (WIN SARS-CoV-2; 30 ± 15%) with those untreated (SARS-CoV-2; 26 ± 12%) (Figs. 2A and 2B).

Since viral infection and replication are correlated but orchestrated by different mechanisms, we asked whether WIN could decrease SARS-CoV-2 replication in hiPSC-CMs. We observed that despite a decrease in average viral titer when comparing SARS-CoV-2 WIN (6.99 × 10⁵ ± 4.39 × 10⁵ PFU/mL) with SARS-CoV-2 (2.18 × 10⁶ ± 9.96 × 10⁵ PFU/mL), the difference was not statistically significant (Fig. 2C).

WIN reduces the secretion of inflammatory cytokines in SARS-CoV-2-infected hiPSC-CMs

The “cytokine storm” is a hallmark of severe COVID-19 cases and cannabinoids have well-known anti-inflammatory properties. We asked whether WIN could reduce the release of the inflammatory cytokines IL-6, IL-8, TNF-α by hiPSC-CMs in vitro. Cells were pretreated with one µM WIN for 24 h, infected for 1 h, and incubated further for 24, 48, and 72 h. Then, the media were harvested at each time point for analysis. We found that cells infected with SARS-CoV-2 released higher levels of cytokines when compared with MOCK, with the exception of IL-8 at 24 and IL-6 at 72 h post-infection (Figs. 3A, 3B and 3C). Most importantly, in all conditions that significantly augmented the release of these pro-inflammatory cytokines, WIN was able to prevent this increase (Figs 3A, 3B and 3C). Of note, whereas the basal amount of cytokines tended to increase during culture time as they accumulated without media changes, WIN did not significantly affect this basal release by comparing MOCK and MOCK WIN groups.

WIN reduces cell death in SARS-CoV-2-infected hiPSC-CMs

It has been previously reported that SARS-CoV-2 infection causes apoptosis in hiPSC-CMs (Perez-Bermejo et al., 2021). As cannabinoids can be protective in some tissues, we investigated whether WIN would protect hiPSC-CMs from cell death. Cells were pretreated with one µM WIN for 24 h, infected for 1 h, and cultivated for additional 24, 48, and 72 h and LDH was measured in the media at these different time points. Forty-eight and 72 h after the infection with SARS-CoV-2 without WIN, the release of LDH increased 463% and 174%, respectively, in hiPSC-CMs. On the other hand, hiPSC-CMs infected with SARS-CoV-2 and exposed to WIN had significantly lower increments of 72% and 40%, respectively (Fig. 3D).