Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility

  1. José Alexandre Salerno
  2. Thayana Torquato
  3. Jairo R. Temerozo
  4. Livia Goto-Silva
  5. Mayara Mendes
  6. Carolina Q. Sacramento
  7. Natalia Fintelman-Rodrigues
  8. Gabriela Vitoria
  9. Leticia Souza
  10. Isis Ornelas
  11. Carla Veríssimo
  12. Karina Karmirian
  13. Carolina Pedrosa
  14. Suelen da Silva Gomes Dias
  15. Vinicius Cardoso Soares
  16. Luiz Guilherme HS Aragão
  17. Teresa Puig-Pijuan
  18. Vinícius W. Salazar
  19. Rafael Dariolli
  20. Diogo Biagi
  21. Daniel Rodrigues Furtado
  22. Helena L. Borges
  23. Patrícia Bozza
  24. Marília Zaluar Guimarães
  25. Thiago Moreno L. Souza
  26. Stevens K. Rehen

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Abstract

Heart dysfunction, represented by conditions such as myocarditis and arrhythmia, has been reported in COVID-19 patients. Therapeutic strategies focused on the cardiovascular system, however, remain scarce. The Sigma-1 receptor (S1R) has been recently proposed as a therapeutic target because its inhibition reduces SARS-CoV-2 replication. To investigate the role of S1R in SARS-CoV-2 infection in the heart, we used human cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CM) as an experimental model. Here we show that the S1R antagonist NE-100 decreases SARS-CoV-2 infection and viral replication in hiPSC-CMs. Also, NE-100 reduces cytokine release and cell death associated with infection. Because S1R is involved in cardiac physiology, we investigated the effects of NE-100 in cardiomyocyte morphology and function. We show that NE-100 compromises cytoskeleton integrity and reduces beating frequency, causing contractile impairment. These results show that targeting S1R to challenge SARS-CoV-2 infection may be a useful therapeutic strategy but its detrimental effects in vivo on cardiac function should not be ignored.

Article activity feed

  1. Rapid Reviews Infectious Diseases

    Kenji Hashimoto

    Review 2: "Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility"

    This preprint uses iPS-derived cardiomyocytes to investigate the role of Sigma-1 Receptor (S1R) during SARS-CoV-2 infection and finds S1R antagonism reduces SARS-CoV-2 replication at the expense of cardiomyocyte function. Reviewers deem these claims reliable.

  2. Rapid Reviews Infectious Diseases

    Carmen Abate

    Review 1: "Inhibition of SARS-CoV-2 infection in human cardiomyocytes by targeting the Sigma-1 receptor disrupts cytoskeleton architecture and contractility"

    This preprint uses iPS-derived cardiomyocytes to investigate the role of Sigma-1 Receptor (S1R) during SARS-CoV-2 infection and finds S1R antagonism reduces SARS-CoV-2 replication at the expense of cardiomyocyte function. Reviewers deem these claims reliable.

  4. ScreenIT

    SciScore for 10.1101/2021.02.20.432092: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    BlindingPlaque numbers were scored in at least 3 replicates per dilution by independent readers blinded to the experimental group and the virus titers were determined by plaque-forming units (PFU) per milliliter. 4.6 Immunocytochemistry and fluorescence image analysis: hiPSC-CMs grown on 96-well plates were fixed using 4% PFA solution (Sigma-Aldrich) for 1 h and stored at 4°C until further processing.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    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).
    anti-TNNT2
    suggested: (Thermo Fisher Scientific Cat# MA5-12960, RRID:AB_11000742)
    anti-OCT4
    suggested: (Thermo Fisher Scientific Cat# MA5-14845, RRID:AB_10979606)
    Primary antibodies were diluted in blocking solution and incubated at 4°C overnight, namely anti-SARS-CoV-2 convalescent serum from a recovered COVID-19 patient (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) and anti-Sigma1R B-5 (1:100, SC-137075 - Santa Cruz Biotechnology).
    anti-SARS-CoV-2
    suggested: None
    anti-SARS-CoV-2 spike protein
    suggested: None
    anti-cardiac troponin T ( cTnT )
    suggested: None
    anti-Sigma1R
    suggested: None
    Next, hiPSC-CMs were washed 3 times with PBS 1X and incubated with the secondary antibodies diluted in blocking solution: goat anti-Human Alexa Fluor 647 (1:400; A-21445 - Invitrogen) and goat anti-Mouse Alexa Fluor 488 (1:400; A-11001 - Invitrogen) for 1 h at room temperature.
    anti-Human Alexa Fluor 647
    suggested: (Molecular Probes Cat# A-21445, RRID:AB_2535862)
    anti-Mouse
    suggested: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)
    Membranes were then incubated overnight at 4°C with primary antibodies (anti-ACE2 (1:1000; MA5-32307 - Thermo Fisher),
    anti-ACE2
    suggested: (Thermo Fisher Scientific Cat# MA5-32307, RRID:AB_2809589)
    anti-Sigma-1 R (1:500; SC-137075 - Santa Cruz Biotechnology), anti-actin (1:2000, MAB1501, Millipore); or anti-GAPDH (1:5000; AM4300 -Thermo Fisher) diluted in TBS-T with 5% non-fat milk.
    anti-GAPDH
    suggested: (Thermo Fisher Scientific Cat# AM4300, RRID:AB_2536381)
    Then, membranes were washed and incubated with peroxidase-conjugated antibodies (goat anti-Mouse IgG (H+L), HRP-conjugate (1:10,000, G21040 -Molecular Probes) and Goat anti-Rabbit IgG (H+L) HRP- conjugate (1:10,000, G21234
    anti-Mouse IgG
    suggested: (Thermo Fisher Scientific Cat# G-21040, RRID:AB_2536527)
    anti-Rabbit IgG
    suggested: (Thermo Fisher Scientific Cat# G-21234, RRID:AB_2536530)
    Experimental Models: Cell Lines
    SentencesResources
    4.4 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 (
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    After 100% confluence was reached, cells were kept in RPMI supplemented with B27 without insulin (both from Thermo Fisher, USA) and with 4 μM CHIR99021 (Merck Millipore Sigma, USA) for one day.
    Merck Millipore Sigma
    suggested: None
    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.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Densitometry analysis was performed using ImageJ software Gel Analysis program and the values obtained represent the ratio of density between the immunodetected protein and the loading control (actin or GAPDH).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Prism v8.0 (GraphPad) was used for data analysis and graphics, where statistical significance was accepted at P<0.05.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04349371TerminatedSaved From COVID-19

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  5. Version published to 10.1101/2021.02.20.432092v1 on bioRxiv