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

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Biochemistry, Biophysics and Molecular Biology

Main article text

 

Introduction

Materials & Methods

hiPSC-CMs culture

Chemicals

Flow cytometry

SARS-CoV-2 propagation

Infections and virus titration

Immunocytochemistry and fluorescence image analysis

Measurements of cell death and cytokines

Gene expression analysis

Protein expression

Beating frequency evaluation

Statistical analysis

Results

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

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

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

S1R inhibition decreases beating in cardiomyocytes

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

Discussion

Conclusions

Supplemental Information

HiPSC-CMs characterization

(A) Flow cytometry data representing the expression of cardiac-specific marker troponin T (cTnT/TNNT2) from four different batches (N = 4), confirming highly efficient differentiation into cardiomyocytes. (B) Immunocytochemistry for cTnT and phalloidin staining for filamentous actin (F-actin). Anti-cTnT (green); phalloidin (red) and nuclei (blue); scale bar = 100 µm. (N = 4; further analyzed in Fig. 4).

DOI: 10.7717/peerj.12595/supp-1

Determination of the staining specificity of the convalescent serum from an in-house patient in comparison with the commercial antibody against the SARS-CoV-2 spike protein (SP)

The CS showed robust and specific immunoreactive signals overlapped with the SP staining in SARS-CoV-2 infected cardiomyocyte cultures (MOI 0.1 at 48 h.p.i), while no such signal was detected in mock-infected condition. The staining was performed in independent experiments (N = 3). Scale bar = 50 µm.

DOI: 10.7717/peerj.12595/supp-2

Cardiac troponin T staining of SARS-CoV-2 infected hiPSC-CMs

Representative immunostaining images of cytoskeleton fragmentation of cardiomyocytes exposed to SARS-CoV-2 (MOI 0.1) for 48 h from 4 independent experiments (N = 4). Anti-convalescent serum (CS) positive staining indicates the presence of SARS-CoV-2 in cells exhibiting Cardiac TnT discontinuity and disruption. cTnT (green), CS (red) and nuclei (blue). Scale bar = 50 µm.

DOI: 10.7717/peerj.12595/supp-3

S1R inhibition does not alter ACE2 expression in hiPSC-CMs

(A) Real-time PCR shows ACE2 mRNA levels. Expression values are normalized by endogenous control genes GAPDH and HPRT1 and are expressed as fold change relative to control (untreated) condition (N = 3). Data are represented as the mean ± S.E.M relative to the untreated condition analyzed by unpaired Welch’s t test (p = 0.0790). (B) Representative blots for ACE2 in protein extracts from untreated hiPSC-CMs or stimulated with 1 µM of NE-100 for 24 h (N = 3). Full-length gels are shown in Fig. S5. (C) Blots performed in samples from three independent batches were quantified by densitometry and normalized by GAPDH expression (N = 3). Data are represented as the mean ± S.E.M relative to the untreated condition analyzed by unpaired Welch’s t test (p = 0.7006). Data points represent independent experiments.

DOI: 10.7717/peerj.12595/supp-4

Full-length agarose gels and Western Blots Membranes

(A) Agarose gel electrophoresis of PCR products amplified from mRNA/cDNA samples obtained from hiPSC-CMs derived from three distinct batches of differentiation (N = 3). Appropriate genomic DNA (gDNA) positive controls and negative no-template controls (NTC) were incorporated into each reaction. The primer pairs used for S1R and GAPDH detection are presented in the methods section. (B-G) Western blot for detection of Sigma-1 receptor (S1R) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (N = 3). (B and E)Image of the full-length membrane used for the detection showing the ladder (Amersham ECL Rainbow Marker - Full range) in the first (B) and second (E) experiments. (C and F) Full-length gel of S1R where the two lanes contain replicates of the detection in samples from two different batches (C) and biological replicates of the detection in one batch (F) (D and G) After S1R detection, membranes were submitted to a stripping protocol as described in the methods section and probed for actin. Cropped versions of the membranes (F) and (G) are represented in the Main Fig. 1 Panel B as representative images of S1R detection. (H–K) Western blot for detection of ACE2 in untreated and NE-100 treated hiPSC-CMs. After transference, the membrane was cut, the upper half was used for ACE2 detection, and the bottom half was used for GAPDH detection. (H, J) Images of the membranes used for detection showing the ladder (Amersham ECL Rainbow Marker - Full range). (I,K) Full-length gels for ACE2 and GAPDH. The lanes used for the representative image in Fig. S4 are highlighted in a box. Other lanes are not related to the experiment described here. The experiment was performed three times, using samples from three different batches (N = 3).

DOI: 10.7717/peerj.12595/supp-5

Untreated hiPSC-CMs beating frequency at baseline, Related to Fig. 4E

DOI: 10.7717/peerj.12595/supp-6

HiPSC-CMs beating frequency at baseline, Related to Fig. 4E

DOI: 10.7717/peerj.12595/supp-7

Untreated hiPSC-CMs beating frequency after 48 h, Related to Fig. 4E

DOI: 10.7717/peerj.12595/supp-8

HiPSC-CMs beating frequency after 48 h of NE-100 exposure, Related to Fig. 4E

DOI: 10.7717/peerj.12595/supp-9

Additional Information and Declarations

Competing Interests

Rafael Dariolli and Diogo Biagi were employed by PluriCell Biotech. The remaining authors declare that they have no competing interests.

Author Contributions

José Alexandre Salerno, Thayana Torquato and Karina Karmirian conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Jairo R. Temerozo, Livia Goto-Silva and Mayara A. Mendes conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Isis M. Ornelas and Carla P. Veríssimo and Teresa Puig-Pijuan performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Vinícius Salazar performed the experiments, analyzed the data, authored or reviewed drafts of the paper, performed software programming and analysis, and approved the final draft.

Rafael Dariolli, Diogo Biagi, Luciana Barreto Chiarini and Stevens K. Rehen conceived and designed the experiments, authored or reviewed drafts of the paper, provided resources, and approved the final draft.

Daniel R. Furtado, Helena L. Borges, Patrícia T. Bozza, Marilia Zaluar P. Guimarães and Thiago M.L. Souza conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Data Availability

The following information was supplied regarding data availability:

Full uncropped gels and membranes and the raw measurements for all results presented in each figure are available in the Supplemental Files.

Funding

This work was supported by intramural grants from D’Or Institute for Research and Education. Students’ scholarships and fellowships were paid by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) or Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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