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

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that lead to damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the “cytokine storm”, strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins six, eight, 18 and tumor necrosis factor-alpha (TNF-α) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.

For many centuries Cannabis sp. has been used for medicinal purposes and, more recently, it has been investigated as a therapeutic agent for cardiovascular diseases (Mendizábal & Adler-Graschinsky, 2007;Pacher et al., 2018). Cannabis has several known compounds, named phytocannabinoids, including delta-9-tetrahydrocannabinol (THC), which is the most abundant and the main psychoactive ingredient, followed in amount by cannabidiol (CBD). Besides phytocannabinoids, there is intensive research on endocannabinoids, such as anandamide and 2-arachidonoylglycerol, and synthetic cannabinoids, such as WIN 55,212-2 (WIN). Nguyen et al. (2021) showed the potential of cannabinoids to decrease SARS-CoV-2 infection, viral replication, and inflammation that are directly related to COVID-19 severity. Treatment with Cannabis extracts decreased ACE2 expression in oral, intestinal, and airway epithelia in vitro . It is noteworthy that cannabinoids have anti-inflammatory properties and exert their biological effect mainly by interaction with the cannabinoid receptors type 1 (CB1) and/or type 2 (CB2), to both of which WIN has high affinity and efficacy (Devane et al., 1988;Munro, Thomas & Abu-Shaar, 1993;Felder et al., 1995;Soethoudt et al., 2017;Sachdev et al., 2019). For instance, WIN was shown to reduce the number of lipopolysaccharide-activated microglia in the brain of an animal model of chronic inflammation (Marchalant, Rosi & Wenk, 2007). Another work showed that WIN decreased TNF-a and IL-6 plasma levels and myeloperoxidase activity in mice with experimental colitis (Feng et al., 2016). An extract fraction from Cannabis sativa Arbel strain enriched in CBD, cannabigerol and tetrahydrocannabivarin presented anti-inflammatory activity in lung epithelial cells treated with TNF-a but another fraction with high CBD containing terpenes in addition to phytocannabinoids enhanced proinflammatory parameters of macrophages (Anil et al., 2021). Additionally, high-CBD Cannabis sativa extracts presented anti-inflammatory properties in the epithelia pretreated with TNF-a and IFN-γ (Lei et al., 2020). Smith, Terminelli & Denhardt (2000) showed that treatment with WIN decreased serum TNF-a and IL-12 and increased IL-10 through the CB1 receptor in mice treated with lipopolysaccharide (Smith, Terminelli & Denhardt, 2000). Investigating the anti-inflammatory potential of Cannabis sativa in cardiomyocytes is important because the "cytokine storm" is a hallmark of COVID-19 and the cardiovascular system is mostly affected in severe cases (Unudurthi et al., 2020). To date, the effects of cannabinoids in human cardiomyocytes infected with SARS-CoV-2 has not been addressed.
In this work, we aimed to investigate the effects of a synthetic cannabinoid, that acts as a mixed CB1/CB2 receptors agonist, in hiPSC-CMs infected by SARS-CoV-2. WIN presented anti-inflammatory and protective properties by reducing the levels of proinflammatory cytokines and cell death in hiPSC-CM but did neither modulate ACE2 nor reduced SARS-CoV-2 infection and replication. Our data suggest that the anti-inflammatory and protective properties of WIN may be used to control inflammation and tissue damage during SARS-CoV-2 infection of heart cells.

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 cm 2 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 × 10 4 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.
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.

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 mM for 72 h. Next, the medium was replaced, cells were washed with PBS 1× and 200 mL 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 mL 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 Ò 200 PRO (Life Sciences, Switzerland) spectrophotometer.

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.

RESULTS
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.

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).

WIN reduces the secretion of inflammatory cytokines in SARS-CoV-2infected 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-a 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).

DISCUSSION
Cannabinoids have been proposed as potential treatment and prevention of COVID-19, due to their antiviral, cytoprotective and anti-inflammatory properties (Marchalant, Rosi & Wenk, 2007;Rossi et al., 2020;Anil et al., 2021). In this study, we showed that the synthetic CB1/CB2 agonist WIN reduced cell damage in SARS-CoV-2infected hiPSC-CMs. Additionally, even though cardiomyocytes are not known for evoking robust inflammatory responses, WIN reduced the release of cytokines by these cells following SARS-CoV-2 infection. To our knowledge, this is the first study showing anti-inflammatory and protective properties of a cannabinoid agonist in hiPSC-CMs infected with SARS-CoV-2. We hypothesized that WIN reduces the levels of ACE2 in hiPSC-CMs, consequently abrogating SARS-CoV-2 infection and viral load in these cells. However, despite a tendency towards increased ACE2 expression in hiPSC-CMs, it was not modulated by WIN in the conditions studied here. ACE2 is downregulated in SARS-CoV-2 infected tissues (Yan et al., 2020;Gheblawi et al., 2020), which is harmful to the heart since ACE2 has a protective role in the cardiovascular system (Huentelman et al., 2005;Zhong et al., 2010). Studies have shown that agonists of cannabinoid receptors, including WIN, cause vasodilation through the activation of CB1 receptors, and are capable of modulating . Cytokine levels were higher in SARS-CoV-2 compared with control (MOCK), and lower in SARS-CoV-2 WIN when compared with SARS-CoV-2. LDH release-absorbance levels relative to MOCK were higher in SARS-CoV-2 compared with SARS-CoV-2 WIN. Data represent means and standard errors of the means (SEM) from three independent experiments from one cell line. Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001, ÃÃÃÃ p < 0.0001.
Full-size  DOI: 10.7717/peerj.12262/ fig-3 vasoactive ligands (Sainz-Cort & Heeroma, 2020;Miklós et al., 2021). One possibility for the tendency towards an increase in the levels of ACE2 in WIN-treated hiPSC-CMs is that this cannabinoid agonist could exert a protective role by preventing receptor downregulation. Although it has been previously shown ) that CBD-rich extracts reduced ACE2 mRNA and protein levels in some epithelia in vitro following TNFa insult, this modulation had not been investigated in SARS-CoV-2 infected cardiomyocytes until now. Despite evidence of cannabinoid receptors expression in murine embryonic stem cells (Jiang et al., 2007) and human cardiomyocytes (Mukhopadhyay et al., 2010), to our knowledge, this is the first description of the expression of CB1 receptor in hiPSC-CMs. The modulation of cannabinoid receptors in cardiomyocytes has also not been explored yet. Our results showed that WIN did not reduce the infection rate or the viral titer in hiPSC-CMs in the conditions studied here. Several studies have examined the effect of cannabinoids on viral infections, especially regarding the role of CB1 and CB2 receptor activation (Reiss, 2010). The CB2 receptor agonist JWH-133 reduced CXCR4-tropic HIV-1 infection of primary CD4+ T cells, whereas the CB1 receptor agonist arachidonoyl-29chloroethylamide had no effect. In another study with HIV-1-infected primary human monocytes, agonists of CB2 receptors limited viral replication (Ramirez et al., 2013). There is still no consensus on the antiviral mechanisms of cannabinoids, however, it is well-known that the selective activation of the CB2 receptor plays a crucial role in the course of viral infection (Rossi et al., 2020). The fact that hiPSC-CMs do not express the CB2 receptor may explain WIN's ineffectiveness in reducing SARS-CoV-2 infection and replication in these cells. Additionally, to date, cannabinoid treatment along with SARS-CoV-2 infection had not been investigated in this cellular model. It is likely that viral infection mechanisms through CB1 and CB2 receptors might vary depending on virus and cell type (Reiss, 2010;Tahamtan et al., 2016).
Although immune cells and cardiac fibroblasts are typically the major players in cytokine production under stressed cardiac conditions (Zhong et al., 2010), cardiomyocytes are also a local source of proinflammatory cytokines (Yamauchi-Takihara et al., 1995;Ancey et al., 2002;Kleinbongard, Schulz & Heusch, 2011;Atefi et al., 2011;Bozzi et al., 2019). In this work, hiPSC-CMs released IL-6, IL-8, and TNF-a at baseline levels and SARS-CoV-2 infection increased all cytokines levels. It has been shown that infection of hiPSC-CMs by Trypanosoma cruzi, the Chagas' disease pathogen, prompted these cells to produce proinflammatory cytokines that caused autocrine cardiomyocyte dysfunction (Bozzi et al., 2019). Cardiac damage in COVID-19 patients can be attributable to hypoxemia due to respiratory dysfunction  but also to the "cytokine storm", which is the uncontrolled systemic inflammatory response likely caused by an imbalance between regulatory and cytotoxic T cells (Meckiff et al., 2020). Even though the "cytokine storm" is one of the hallmarks of SARS-CoV-2 infection (Coperchini et al., 2020), one cannot rule out that cytokines locally released contribute to tissue damage, as seen, for example, in Trypanosoma cruzi cardiac infection (Bozzi et al., 2019). Here we found that WIN decreased the levels of IL-6, IL-8, and TNF-a released by SARS-CoV-2-infected hiPSC-CMs. An in vitro study of cortical astrocytes treated with Amyloid-β 1-42 , which is a neurotoxic protein, showed that WIN reduced TNF-a and IL-1β levels, while preventing cell death (Aguirre-Rueda et al., 2015). In another study, WIN decreased the activity of peroxisome proliferator-activated receptor alpha and TNF-a levels in the heart tissue of mice with cardiac dysfunction (Rahmatollahi et al., 2016), reinforcing its anti-inflammatory and protective properties in cardiac tissue. THC and WIN are structurally different and accordingly have different efficacies towards activation of cannabinoid signaling pathways (Soethoudt et al., 2017). However, they are both mutual CB1 and CB2 receptor agonists (Compton et al., 1992) and can produce similar pharmacological effects, depending on the assay (Fan et al., 1994). THC presented a protective role against hypoxia in neonatal murine cardiomyocytes by reducing the levels of LDH (Shmist et al., 2006). Since neonatal murine cardiomyocytes expressed CB2, but not CB1, the authors suggest that cardioprotection provided by THC occurs via the CB2 receptor. Herein, we were able to show that hiPSC-CMs expressed CB1 but not CB2. In human cardiomyocytes, WIN decreased the release of LDH release and this effect could be mediated by CB1, to which WIN has high affinity. Other receptor candidates, such as transient receptor potential vanilloid (TRPV) channels (Freichel et al., 2017) can not be discarded. Nonetheless, the modulation of TRPV channels by WIN occurs at ten µM or higher (Jeske et al., 2006;Koch et al., 2011), which are, at least, ten times above the concentration used in our study.

CONCLUSION
This study showed that pretreatment with a cannabinoid receptor agonist reduced cytotoxicity and the release of proinflammatory cytokines by human cardiomyocytes infected with SARS-CoV-2. These results suggest that the therapeutic potential of cannabinoids in protecting the heart against SARS-CoV-2 infection should be further explored, in particular regarding selective action on the CB1 receptor.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This work was supported by the National Council of Scientific and Technological Development (CNPq) (Grant Number: 313688/2020-6) and scholarship grants from Coordination for the Improvement of Higher Education Personnel (CAPES) and CNPq. Intramural grants were provided from the D'Or Institute for Research and Education (IDOR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: National Council of Scientific and Technological Development (CNPq): 313688/2020-6. D'Or Institute for Research and Education (IDOR).
Diogo Biagi performed the experiments, authored or reviewed drafts of the paper, and approved the final draft. Estela M. Cruvinel performed the experiments, authored or reviewed drafts of the paper, and approved the final draft. Rafael Dariolli performed the experiments, authored or reviewed drafts of the paper, and approved the final draft. Daniel R. Furtado analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. Patrícia T. Bozza analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. Helena L. Borges analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. Thiago M. L. Souza analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. Marília Zaluar P. Guimarães conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. Stevens K. Rehen conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Human Ethics
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): This work was approved by the Ethics committee of D'Or Institute of Research and Education (IDOR) (39474020.8.0000.5249).

Data Availability
The following information was supplied regarding data availability: The raw data is available as a Supplementary File.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.12262#supplemental-information.