Inhibition of SARS-CoV-2 infection in human iPSC-derived cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoarchitecture and beating

hiPSC-CMs culture

Fresh human iPSCs-derived cardiomyocytes were purchased from Pluricell (São Paulo, Brazil) and the protocol for cardiomyocyte differentiation is described (Cruvinel et al., 2020). Upon arrival, cardiomyocytes were allowed to regain contractility and maintained at 37 °C in a humidified atmosphere with 5% CO₂. Cardiomyocytes were used between days 30 to 40 of differentiation.

Chemicals

4-Methoxy-3-(2-phenylethoxy)-N,N-dipropylbenzeneethanamine hydrochloride (NE-100 hydrochloride) was purchased from Tocris (3133). Stock and work solutions were prepared using 100% dimethyl sulfoxide sterile-filtered (DMSO; D2650 - Sigma-Aldrich).

Flow cytometry

Cardiomyocytes were plated on 6-well plates coated with GELTREX and cultivated for 7 days. After cell dissociation, cells were fixed with 1% paraformaldehyde (PFA), permeabilized with Triton 0.1% (Sigma Aldrich) and Saponin 0.1% (Sigma Aldrich) and stained with the antibodies anti-TNNT2 (1:2500; Thermo Fisher, MA5-12960) and anti-OCT4 (1:200, Thermo Fisher, MA5-14845). FC data was acquired using a Canto BD flow cytometer for each batch of differentiation and analyzed using the FlowJo Software considering 1%–2% of false-positive events.

SARS-CoV-2 propagation

SARS-CoV-2 was expanded in Vero E6 cells from an isolate obtained from a nasopharyngeal swab of a confirmed case in Rio de Janeiro, Brazil (GenBank accession no. MT710714). The National Review Board approved the study protocol (CONEP 30650420.4.1001.0008) for clinical samples, and informed consent was obtained from all participants or patients’ representatives. Viral isolation was performed after a single passage in cell culture in 150 cm² flasks with high glucose DMEM 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), and virus stocks were kept in −80 °C ultra-low temperature freezers.

Infections and virus titration

Cardiomyocytes were infected with SARS-CoV-2 at MOI of 0.1 in CDM3 media without serum. After 1 h, cells were washed and incubated with complete medium with treatments or not. For virus titration, monolayers of Vero E6 cells (2 × 10⁴ cells/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, 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 PFU per milliliter (PFU/ml).

Immunocytochemistry and fluorescence image analysis

Cells were fixed using 4% PFA solution (Sigma-Aldrich) for 1 h, washed with 1X PBS and then incubated with permeabilization/blocking solution (0.3% Triton X-100/3% bovine serum albumin) for 1 h. Primary antibodies were diluted in blocking solution and incubated at 4 °C overnight, namely anti-SARS-CoV-2 convalescent serum (1:1000); anti-SARS-CoV-2 spike protein monoclonal antibody (SP) (1:500, G632604; Genetex); anti-cardiac troponin T (cTnT) (1:500, MA5-12960; Invitrogen), anti-VDAC1/Porin (1:100, ab34726; Abcam), anti-Calnexin (1:50, #2433; Cell Signaling Technology) and anti-Sigma1R B-5 (1:100, SC-137075; Santa Cruz Biotechnology). The use of the convalescent serum from COVID-19 patients was approved by CAAE number: 30650420.4.1001.0008. Next, hiPSC-CMs were washed with PBS 1X and incubated with the secondary antibodies diluted in blocking solution: goat anti-Human Alexa Fluor 647 (1:400; A-21445; Invitrogen), goat anti-Rabbit Alexa Fluor 546 and goat anti-Mouse Alexa Fluor 488 (1:400; A-11001; Invitrogen) for 1 h at room temperature. Actin filaments were stained with Alexa Fluor 568 phalloidin (1:10; A-12380; Life Technologies) for 1 h. Nuclei were stained with 300 nM 4′-6-diamino-2-phenylindole (DAPI) for 5 min and each well was mounted with 50% PBS-Glycerol.

For quantitative analysis, images were acquired using Operetta^® High-Content Screening System (Perkin Elmer) with a 20x long working distance (WD) objective lens from at least 10 fields per well. For cell surface area measurement, images of F-actin stained cardiomyocytes were segmented using Cellpose and the area was measured using NIH ImageJ software (Stringer et al., 2021). For the other analyses, data were evaluated using the Columbus™ Image Data Storage and Analysis System (Perkin Elmer) for image segmentation and object detection. Fluorescence threshold was set to determine positive and negative cells for each marker. Representative immunostaining images were acquired on a Leica TCS-SP8 confocal microscope using a 63x oil-immersion objective lens.

Measurements of cell death and cytokines

Monolayers of hiPSC-CMs in 96-well plates (70–90% confluence) were treated with various concentrations of NE-100. The neutral red assay was performed, and viability was estimated by the percentage relative to untreated condition (vehicle) using the mean of 6 technical replicates per experiment.

The levels of IL-6, IL-8, and TNF-α were quantified in the supernatants from uninfected and SARS-CoV-2-infected hiPSC-CMs by ELISA (R&D Systems), following manufacturer’s instructions. Control groups (mock and cells infected with SARS-CoV-2 only) were also analyzed in Aragão et al. (2021). The results were obtained as picograms per milliliter (pg/ml) and are expressed as fold-change relative to untreated uninfected control. 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. Supernatants were centrifuged at 5,000 rpm for 1 min, to remove cellular debris.

Gene expression analysis

Total RNA was isolated using TRIzol, according to the manufacturer’s recommendations (Thermo Fisher Scientific) and digested using DNase I, Amplification Grade (Invitrogen), following the manufacturer’s instructions. DNase-treated RNA samples (1 µg) were converted to complementary DNA (cDNA) using the M-MLV enzyme (Thermo Fisher Scientific). Qualitative endpoint PCR reactions were executed with the following primer sequences: S1R (forward 5′- AGTAGGACCATGCACTCACACC-3′; reverse: 5′- CCCCATCCTTAACTCTAGAACC-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 10 µL mixture containing 0.25 U GoTaq G2 Hot Start Polymerase (Promega), 1x GoTaq G2 Buffer, 1.5 mM MgCl₂ (Invitrogen), 200 nM of each primer (forward and reverse), 200 µM dNTP mixture containing the four deoxyribonucleotides (dATP, dCTP, dTTP, and dGTP), and 10 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, 58 °C for 15 s and 72 °C for 15 s using the ProFlex™ PCR System Thermal Cycler (Applied Biosystems). Subsequently, total amount of PCR product was separated by electrophoresis at 120 V for 30 min in 2% agarose gel diluted in 1X Tris-acetate EDTA buffer (w/v) and stained with 0.01% of SYBR Safe (Thermo Fisher).

For real-time quantitative PCR, the reactions were conducted in three replicates with a final volume of 10  µL containing 1X GoTaq qPCR Master Mix (Promega), 300 nM CXR Reference Dye, 200 nM of each SYBR green designed primers: Angiotensin I Converting Enzyme 2 (ACE2; forward: 5′-CGAAGCCGAAGACCTGTTCTA-3′; reverse: 5′-GGGCAAGTGTGGACTGTTCC-3′); Natriuretic Peptide A (ANP; forward: 5′-CAACGCAGACCTGATGGATTT-3′; reverse: 5′-AGCCCCCGCTTCTTCATTC-3′); Spliced X-Box Binding Protein 1 (sXBP1; forward: 5′- CTGAGTCCGAATCAGGTGCAG-3′; reverse: 5′-ATCCATGGGGAGATGTTCTGG-3′); Unspliced X-Box Binding Protein 1 (usXBP1; forward: 5′-CAGCACTCAGAC TACGTGCA-3′; reverse: 5′- ATCCATGGGGAGATGTTCTGG-3′); Total X-Box Binding Protein 1 (totalXBP1; forward: 5′- TGGCCGGGTCTGCTGAGTCCG-3′; reverse: 5′-ATCCATGGGGAGATGTTCTGG-3′); C/EBP-homologous protein (CHOP; forward: 5′-AGAACCAGGAAACGGAAACAGA-3′; reverse: 5′-TCTCCTTCATGCGCTGCTTT-3′); Actinin Alpha 1 (ACTN1; forward: 5′-CCCGAGCTGATTGACTACGG-3′; reverse: 5′-GCAGTTCCAACGATGTCTTCG-3′); Actinin Alpha 2 (ACTN2; forward: 5′-GACATCGTGAACACCCCTAAAC-3′; reverse: 5′- CCGCAAAAGCGTGGTAGAA); Actin Alpha 1 (ACTA1; forward: 5′-TGCCAACAACGTCATGTCG-3′; reverse: 5′-CAGCGCGGTGATCTCTTTCT-3′); Troponin I3, Cardiac Type (TNNI3; forward: 5′-TTTGACCTTCGAGGCAAGTTT-3′; reverse: 5′-CCCGGTTTTCCTTCTCGGTG-3′); Troponin I1, Slow Skeletal Type (TNNT1; forward: 5′-TGATCCCGCCAAAGATCCC-3′; reverse: 5′-TCTTCCGCTGCTCGAAATGTA-3′); Myosin Heavy Chain 6 (MYH6; forward: 5′- GCCCTTTGACATTCGCACTG-3′; reverse: 5′-GGTTTCAGCAATGACCTTGCC-3′); Myosin Heavy Chain 7 (MYH7; forward: 5′-TCACCAACAACCCCTACGATT-3′; reverse: 5′- CTCCTCAGCGTCATCAATGGA-3′); and 10 ng of cDNA per reaction. The reactions were performed on a StepOnePlus ™ Real-Time PCR System thermocycler (Applied Biosystems). The relative expression of the genes of interest (GOI) was normalized by endogenous control genes: Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH; forward: 5′-GCCCTCAACGACCACTTTG-3′; reverse: 5′-CCACCACCCTGTTGCTGTAG-3′) and Hypoxanthine Phosphoribosyl transferase 1 (HPRT-1; forward 5′-CGTCGTGATTAGTGATGATGAACC-3′; reverse: 5′-AGAGGGCTACAATGTGATGGC-3′).

Data analysis was performed with the N₀ method implemented in LinRegPCR v. 2020.0 software, which considers efficiency estimated by the window-of-linearity method, as proposed by Ramakers et al. (2003), Ruijter et al. (2009). N₀ values were calculated using default parameters and the arithmetic mean of N₀ values from GOI were normalized by taking its ratio to the N₀ of the geometric mean of the endogenous control genes (REF) GAPDH and HRRT-1 (N₀GOI /N₀REF).

Protein expression

Media was completely removed from hiPSC-CMs, and 40 µL of sample buffer without bromophenol blue (62.5 mM Tris–HCl, pH 6.8, containing 10% glycerol, 2% SDS, and 5% 2-mercaptoethanol) was added to each well. Next, cell extracts were boiled at 95 °C for 5 min, centrifuged 16,000 x g for 15 min at 4 °C, and the supernatant was collected. Protein content was estimated using the Bio-Rad Protein Assay (#5000006; Bio-Rad). After the addition of bromophenol blue (0.02%), samples were separated by electrophoresis on an 8% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes.

Western blotting was carried out with minor modifications from the described (Towbin, Gordon & Staehelin, 1989). Briefly, membranes were blocked in 5% non-fat milk diluted in Tris-Buffered Saline with 0.1% Tween-20 (TBS-T) for 1 h at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies diluted in TBS-T with 5% non-fat milk. Then, membranes were washed and incubated with peroxidase-conjugated antibodies. The signals were developed using ECL Prime Western Blotting System (#GERPN2232, Sigma) for 5 min and chemiluminescence was detected with an Odyssey-FC System^® (LI-COR Biosciences).

Stripping protocol was performed to break bonds between the antibodies and the transferred proteins in order to reuse membranes. Briefly, membranes were incubated for three cycles of 10 min in a stripping buffer (pH 2.2, 200 mM glycine, SDS 0.1% and 1% Tween-20). Then, the buffer was discarded, the membranes were washed for 5 min with PBS and 5 min with 0.1% TBS-T (three times each). Next, membranes were blocked again and proceeded with the above-described steps. Full-length membranes are displayed in Fig. S5.

Beating frequency evaluation

hiPSC-CMs were plated on 96-well plates (1.8 ×10⁴ cells per well) and treated with NE-100 1 µM or vehicle (DMSO) before incubating at 37 °C. At 24- and 48-hours post-treatment, beating frequency was measured by manually counting the synchronous contractions of the monolayer for 60 s (beats per minute). Cells were observed using an EVOS cell imaging system (Thermo Fisher Scientific), in brightfield mode. Representative videos were recorded using Operetta^® High-Content Screening System (Perkin Elmer) for each condition at baseline and 48 h after treatment with vehicle or NE-100.

Statistical analysis

Data are presented as mean values, and error bars indicate the standard error of the mean (S.E.M). Statistical differences were analyzed using nested t-test, unpaired two-tailed Welch’s t-test and between three or more groups, unpaired multiple t-tests (Holm-Sidak method) or ordinary one-way ANOVA with Holm-Sidak post-hoc. Prism v8.0 (GraphPad) was used for data analysis and graphics, where statistical significance was accepted at P < 0.05. The tests and p values are specified in figure legends and the symbols represent p < 0.05, ** p < 0.01, * p < 0.001, *** p < 0.0001.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) express the Sigma-1 receptor (S1R)

Human cardiomyocytes were differentiated from induced pluripotent stem cells according to established protocols (Cruvinel et al., 2020). Differentiation to cardiomyocytes resulted in low expression of the pluripotency marker OCT-4 (0.8% ± 0.4%) and high expression of the specific cardiac muscle marker troponin T (cTnT) (88.4% ± 8.4%) (Fig. S1A). The presence of cTnT was confirmed by immunostaining, as was cell morphology with F-actin staining (Fig. S1B).

Previous studies showed that S1R was expressed in the human heart tissue and in atrial and ventricular cardiomyocytes of rodents (Novakova et al., 1995; Novakova et al., 2010; Fagerberg et al., 2014; Abdullah et al., 2020). Here, we showed S1R mRNA and protein in hiPSC-CMs (Figs. 1A and 1B, respectively). Immunostaining showed that S1R is in the nuclei and in the perinuclear region of the cells (Fig. 1C). This is consistent with previous findings of the distribution of S1R in cells from rodents and yeasts and in human cell lines (Dussossoy et al., 1999; Hayashi & Su, 2003a). S1R is enriched at the mitochondria-associated ER membrane (MAM) (Hayashi & Su, 2007; Morales-Lázaro, González-Ramírez & Rosenbaum, 2019). We found that part of cytosolic S1R staining was in close apposition with the mitochondrial marker VDAC1/Porin (Fig. 1D) and with the ER chaperone lectin Calnexin (CNX) (Fig. 1E) in hiPSC-CMs.

Inhibition of S1R reduces infection and replication of SARS-CoV-2 and prevents cell death in human cardiomyocytes

To test whether S1R had a direct influence on SARS-CoV-2 infection in human cardiomyocytes, the selective S1R antagonist, NE-100, was used to inhibit this receptor in hiPSC-CMs prior to exposure to the virus. NE-100 has a binding affinity for S1R within the nanomolar range and an IC50 200 times higher for other receptors, such as dopamine, serotonin, and phencyclidine receptors (Okuyama et al., 1993). Exposure of cardiomyocytes to NE-100 in the micromolar range for 24 h was reported to abolish the effects of a S1R agonist, similarly to what was observed using small interference RNA (siRNA) (Tagashira et al., 2013). Then, following 24 h exposure to 1 µM NE-100, hiPSC-CMs were infected with SARS-CoV-2 at the multiplicity of infection (MOI) 0.1. Immunofluorescence using convalescent serum (CS) from a recovered COVID-19 patient showed that 57.3% (±11.1%) of cells were infected at 48 h.p.i (hours post-infection) (Fig. 2A). CS-staining was confirmed with co-localization with the specific SARS-CoV-2 Spike Protein (SP) (Fig. S2).

Importantly, the inhibition of S1R reduced the percentage of infected hiPSC-CMs to 35.8% (±2.5%) (Figs. 2A and 2B). Moreover, whereas infection of hiPSC-CMs with SARS-CoV-2 led to the production of infectious virions measured in the plaque assay, exposure to NE-100 considerably diminished this viral yield, with an average reduction of 82% plaque forming units (PFU) at 48 h.p.i (Fig. 2C). SARS-CoV-2 infection was previously shown to induce cytopathic features in hiPSC-CMs, mostly related to myofibrillar disruption, as described by other groups (Perez-Bermejo et al., 2021; Marchiano et al., 2021). Likewise, we detected a pattern of structural disarrangement of cardiac troponin T staining, as shown in Fig. S3.

The release of lactate dehydrogenase (LDH) confirmed that SARS-CoV-2 caused cardiomyocyte death, as previously reported (Sharma et al., 2020). At 24, 48, and 72 h.p.i, LDH was elevated by 2.3, 5.6 and 2.7-fold, respectively, in SARS-CoV-2-infected cells when compared to controls (Fig. 2D). NE-100 was able to decrease the release of LDH at 48 h.p.i and also tended to decrease cytotoxicity at 72 h.p.i (Fig. 2D). There was no increase in the release of LDH when comparing uninfected to NE-100 treated controls (Fig. 2D). These results suggest that the inhibition of S1R does not induce cell death and may protect hiPSC-CMs by decreasing infection.

S1R is engaged in ER protein synthesis and acts as a chaperone for proteins translocating to cell surface (Vela, 2020). We investigated whether the inhibition of S1R could be related to modifications in the host cell receptor for viral entry, since it partially prevented the infection. The expression of the main viral entry receptor, ACE2, was measured after 24 h exposure to 1 µM NE-100. There was a reduction in the levels of ACE2 mRNA, albeit not statistically significant (p = 0.0790) (Fig. S4A). However, western blot analysis of ACE2 after NE-100 treatment did not show differences at the protein level (Figs. S4B and S4C). These data suggest that the inhibition of SARS-CoV-2 infection in hiPSC-CMs can occur through mechanisms other than a reduction in the availability of ACE2 under the above-described conditions.

NE-100 attenuates cytokine release by SARS-CoV-2 infected hiPSC-CMs

Cell death is the final denouement of hiPSC-CMs at 72 h.p.i (Sharma et al., 2020; Aragão et al., 2021). To investigate events before cell death, we analyzed some cytokines associated with COVID-19 at 24 and 48 h.p.i. This strategy was previously shown to be significant in the context of the infection of hiPSC-CMs by other pathogens that target the heart (Bozzi et al., 2019).

SARS-CoV-2 infection increased the release of interleukin (IL)-6 when compared to control. NE-100 decreased this response at 24 h.p.i and 48 h.p.i (Figs. 3A and 3B). Conversely, no differences in the secretion of IL-8 were observed at 24 h.p.i (Fig. 3A), however, at 48 h.p.i, SARS-CoV-2 increased IL-8 by 5.5-fold relative to the mock condition (Fig. 3B). Comparably to the modulation of IL-6, NE-100 decreased the secretion of IL-8 (Fig. 3B). Tumor necrosis factor-alpha (TNF-α) levels were only transiently increased at 24 h.p.i (3.3-fold) but were nevertheless inhibited by previous NE-100 treatment (Fig. 3B).

S1R inhibition decreases beating in cardiomyocytes

To investigate the consequences of the inhibition of S1R in human cardiomyocytes by itself, we conducted further tests on the effects of NE-100 in hiPSC-CMs. We first analyzed whether NE-100 induces cell death. NE-100 does not decrease cell viability after 72 h at concentrations ranging from 10 nM to 10 µM as measured by the neutral red assay (Fig. 4A). Also, hiPSC-CMs treated with 1 µM NE-100 for 48 h showed no changes in the number of pyknotic nuclei, which represents an irreversible chromatin-condensed nuclear state characteristic of cell death (Fig. 4B).

Furthermore, given that S1R function is important for the ER stress response, we investigated whether NE-100 modulates the endonuclease activity of inositol-requiring enzyme 1α (IRE1α) by analysis of splicing of XBP1 mRNA, measuring the levels of spliced XBP1 mRNA (sXBP1), in addition to the unspliced (uXBP1) and total XBP1 mRNAs (Oslowski & Urano, 2011). We found low levels of sXBP1 mRNA and high levels of uXBP1 mRNA in hiPSC-CMs which are consistent with cells under non-ER stress condition (Fig. 4C). NE-100 treatment did not affect levels of sXBP1 or uXBP1 mRNAs/total XBP1 (Fig. 4C). These results indicate that NE-100 did not induce activation of IRE1α in hiPSC-CMs under these conditions. Another indicator of ER stress response is the upregulation of the C/EBP-homologous protein (CHOP or GADD153). Again, we did not find changes in CHOP mRNA after NE-100 treatment (Fig. 4D).

These data suggest that NE-100 does not induce hiPSC-CMs death or ER stress response in the absence of additional stressor stimuli. However, one of the main functional features displayed by cultured cardiomyocytes is their ability to perform spontaneous contractions. In that matter, hiPSC-CMs are suitable to evaluate drug-induced changes in contractility (Pointon et al., 2015; Niehoff et al., 2019). We observed a decrease from 17.7 (±5.9) beats per minute (bpm) to 6.7 (±1.8) bpm and from 14.7 (±2.8) bpm to 4.1 (±0.8) bpm after 24 h and 48 h of NE-100 exposure, respectively (Fig. 4E and Videos S1–S4). These results suggest that the inhibition of S1R leads to changes in beating rate and a putative deviation in contractility.

The S1R antagonist NE-100 induces maladaptive transcriptional and structural changes in human cardiomyocytes

The findings on the decremental impact of NE-100 on the cardiomyocytes beating rate led us to pursue this issue further. S1R plays a cardioprotective role during maladaptive cardiac remodeling and the anti-hypertrophic properties of S1R agonists have been described (Tagashira & Fukunaga, 2012; Tagashira et al., 2013; Hirano, Tagashira & Fukunaga, 2014). Therefore, we also investigated if the S1R antagonist NE-100 alone could induce a hypertrophic response. To that end, we quantified the expression of key genes upon S1R inhibition. First, by measuring mRNA levels of the atrial natriuretic peptide (ANP), which is one of the main transcripts of the fetal program for cardiac growth. ANP overexpression is frequently correlated to cardiac hypertrophy but NE-100 decreased ANP mRNA expression by 1.49-fold after 24 h (Fig. 5A). There was also no increase in the average cell surface area of hiPSC-CMs observed by F-actin staining. In fact, more like the contrary, NE-100 exposure caused a decrease in the average cell surface area after 48 h (Fig. 5B), which is a phenotype opposite to the expected for cardiomyocyte hypertrophic responses in vitro (Watkins et al., 2012).

After observing cell shrinking in longer exposures to NE-100, we then hypothesized that S1R inhibition could be triggering morphological changes. Hence, we investigated the expression of myofibril-associated genes important for cytoarchitecture maintenance and observed a downregulation of 4 out of 7 genes investigated. The decreased expression of ACTN2 (−1.53-fold), ACTA1 (−11.1-fold), TNNI3 (−2.32-fold) and TNNT1 (−2.43-fold) after the inhibition of S1R suggests a transcriptional regulation that could impair sarcomeric organization and cardiomyocyte cytoskeletal integrity (Fig. 5C). ACTN1 and MYH7 expression did not show significant changes upon NE-100 exposure (Fig. 5C). Interestingly, MYH7 upregulation is highly associated with hypertrophy (Krenz & Robbins, 2004; Gupta, 2007). Therefore, this result corroborates the assumption that the inhibition of S1R in the absence of a hypertrophic stimulus is not sufficient to activate the molecular profile of cardiac hypertrophy in hiPSC-CMs (i.e., ANP and MYH7 upregulation). Intriguingly, MYH6 was the only transcript upregulated by 3.44-fold (p = 0.0502) (Fig. 5C), which could represent a transcriptional compensatory adaptation.

Furthermore, cardiac troponin T (cTnT) staining analysis revealed a significant decrease both in immunoreactive area and fluorescence intensity on hiPSC-CMs exposed to NE-100 when compared to untreated controls (Figs. 5D and 5E, respectively). These results are probably attributable also to a decrease in cellular cTnT content rather than exclusively to a change in the cell area, indicating a considerable loss of troponin. Consistently, the morphology of cytoskeletal fibers was grossly affected after 48 h, as shown in confocal fluorescence images (Fig. 5F). Taken together, our results suggest that S1R inhibition caused neither change in cell viability nor induction of hypertrophy parameters. However, despite this apparent lack of negative effects related to cell death and hypertrophy, there was a downregulation of genes encoding structural proteins and cytoskeletal impairment via a decrease in cardiac troponin T content. The latter significant effects could explain in part the beating frequency reduction of human cardiomyocytes exposed to NE-100.