論文の公開元へSARS-CoV-2 spike receptor-binding domain is internalized and promotes protein ISGylation in human induced pluripotent stem cell-derived cardiomyocytes
概要
Title
SARS-CoV-2 spike receptor-binding domain is
internalized and promotes protein ISGylation in
human induced pluripotent stem cell-derived
cardiomyocytes
Author(s)
Okuno, Shota; Higo, Shuichiro; Kondo, Takumi et
al.
Citation
Scientific Reports. 2023, 13(1), p. 21397
Version Type VoR
URL
rights
https://hdl.handle.net/11094/93523
This article is licensed under a Creative
Commons Attribution 4.0 International License.
Note
Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University
www.nature.com/scientificreports
OPEN
SARS‑CoV‑2 spike receptor‑binding
domain is internalized
and promotes protein ISGylation
in human induced pluripotent stem
cell‑derived cardiomyocytes
Shota Okuno 1, Shuichiro Higo 1,2*, Takumi Kondo 1, Mikio Shiba 1, Satoshi Kameda 1,
Hiroyuki Inoue 1, Tomoka Tabata 1, Shou Ogawa 1, Yu Morishita 1, Congcong Sun 1, Saki Ishino 3,
Tomoyuki Honda 4,5, Shigeru Miyagawa 6 & Yasushi Sakata 1
Although an increased risk of myocarditis has been observed after vaccination with mRNA encoding
severe acute respiratory syndrome coronavirus 2 spike protein, its underlying mechanism has
not been elucidated. This study investigated the direct effects of spike receptor-binding domain
(S-RBD) on human cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs).
Immunostaining experiments using ACE2 wild-type (WT) and knockout (KO) iPSC-CMs treated with
purified S-RBD demonstrated that S-RBD was bound to ACE2 and internalized into the subcellular
space in the iPSC-CMs, depending on ACE2. Immunostaining combined with live cell imaging using
a recombinant S-RBD fused to the superfolder GFP (S-RBD-sfGFP) demonstrated that S-RBD was
bound to the cell membrane, co-localized with RAB5A, and then delivered from the endosomes to
the lysosomes in iPSC-CMs. Quantitative PCR array analysis followed by single cell RNA sequence
analysis clarified that S-RBD-sfGFP treatment significantly upregulated the NF-kβ pathway-related
gene (CXCL1) in the differentiated non-cardiomyocytes, while upregulated interferon (IFN)-responsive
genes (IFI6, ISG15, and IFITM3) in the matured cardiomyocytes. S-RBD-sfGFP treatment promoted
protein ISGylation, an ISG15-mediated post-translational modification in ACE2-WT-iPSC-CMs,
which was suppressed in ACE2-KO-iPSC-CMs. Our experimental study demonstrates that S-RBD
is internalized through the endolysosomal pathway, which upregulates IFN-responsive genes and
promotes ISGylation in the iPSC-CMs.
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARSCoV-2), has been declared a pandemic and poses a serious threat to public health1,2. Vaccination is the cornerstone of pandemic control, and COVID-19 vaccines using various platforms, including a new mRNA vaccine, have been developed with an unprecedented speed3,4. COVID-19 mRNA vaccines encoding SARS-CoV-2
spike proteins exhibit remarkable e ffectiveness5–7. However, after COVID-19 vaccination began in the general
population, numerous case reports of myocarditis after the administration of COVID-19 vaccines have been
published8–12, and a significant association between myocarditis and mRNA vaccines has been reported in a few
observational studies13–17. COVID-19 vaccine-associated myocarditis is an important issue.
However, the mechanisms underlying myocarditis after COVID-19 vaccination are poorly understood.
Although several mechanisms, including molecular mimicry or T-cell involvement by adaptive immunity,
1
Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565‑0871,
Japan. 2Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine,
Suita, Osaka 565‑0871, Japan. 3CoMIT Omics Center, Osaka University Graduate School of Medicine, Suita,
Osaka 565‑0871, Japan. 4Department of Virology, Okayama University Graduate School of Medicine, Dentistry
and Pharmaceutical Sciences, Kita‑Ku, Okayama 700‑8558, Japan. 5Department of Virology, Faculty of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama University, Kita‑Ku, Okayama 700‑8558, Japan. 6Department
of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka 565‑0871, Japan. *email:
higo-s@cardiology.med.osaka-u.ac.jp
Scientific Reports |
(2023) 13:21397
| https://doi.org/10.1038/s41598-023-48084-7
1
Vol.:(0123456789)
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a
iPSCs
2
SARS-CoV-2
SS-RBD
iPSC-CMs
48 h
RPMI
ascorbic acid+albumin
Day 0
b
kDa
Immunostaining
Western blot
Wnt-C59
XAV-939
CHIR
Day 2
Day 4
iPSCs
iPSC-CMs
c
Hoechst / ACE2
150-
ACE2
Day 28
Day 14
Replating
Hoechst / Troponin T
100-
α-actinin
100-
GAPDH
37-
d
50 μm
ACE2
e
S-RBD-His
ACE2
S-RBD-His
F
50 μm
50 μm
ACE2 / S-RBD-His
ACE2 / S-RBD-His
f
kDa
WT
KO
Relative expression
100-
α-actinin
h
Hoechst / ACE2
Hoechst / Troponin T
***
150-
ACE2
g
100-
WT
50 μm
KO
37-
GAPDH
50 μm
i
ACE2
50 μm
Scientific Reports |
Vol:.(1234567890)
(2023) 13:21397 |
S-RBD-His
ACE2 / S-RBD-His
j
ACE2
S-RBD-His
ACE2 / S-RBD-His
50 μm
https://doi.org/10.1038/s41598-023-48084-7
2
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◂Figure 1. Internalization of SARS-CoV-2 S-RBD/ACE2 complex into the human iPSC-CMs. (a) Time course
of monolayer differentiation into the cardiomyocytes. Differentiated cardiomyocytes were replated on day
14 after differentiation for further analysis. The iPSC-CMs were incubated with purified His-tagged SARSCoV-2 S-RBD protein for 48 h before immunostaining and western blotting on day 28 after differentiation. (b)
Whole cell lysates were extracted from the iPSCs and iPSC-CMs on day 28 after differentiation and analyzed by
western blotting using the indicated antibodies. Original blots are presented in Supplementary Fig. S5. (c) The
iPSC-CMs were fixed and immunostained with the indicated antibodies on day 28 after differentiation. Nuclei
were detected by Hoechst staining. (d) The iPSC-CMs were incubated with 1250 ng/mL purified His-tagged
SARS-CoV-2 S-RBD protein for 48 h before immunostaining on day 28 after differentiation with the indicated
antibodies. White arrows show the accumulation of S-RBD at the periphery of the iPSC-CMs co-localized with
ACE2. (e) The iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD protein for
48 h before immunostaining on day 28 after differentiation with the indicated antibodies. Areas enclosed within
the white squares are enlarged at the bottom. White arrows indicate the S-RBD/ACE2 complex internalized in
the subcellular space. (f) Whole cell lysates were extracted from ACE2-WT-iPSC-CMs and ACE2-KO-iPSCCMs on day 28 after differentiation and analyzed by western blotting using the indicated antibodies. Original
blots are presented in Supplementary Fig. S5. (g) Quantified ACE2 protein expression levels were normalized
by GAPDH expression in ACE2-WT-iPSC-CMs and ACE2-KO-iPSC-CMs (n = 4). Data are presented as the
mean ± SD. Statistical differences were calculated using Student’s t-test. ***p < 0.001. (h) ACE2-WT-iPSC-CMs
and ACE2-KO-iPSC-CMs were fixed and immunostained on day 28 after differentiation using the indicated
antibodies. (i) ACE2-WT-iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD
protein for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies. (j) ACE2KO-iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD protein for 48 h
before immunostaining on day 28 after differentiation with the indicated antibodies.
have been proposed; one possible hypothesis is that free-floating spike protein or its subunits/peptide fragments after proteolytic cleavage directly affect the cardiomyocytes by changing the gene expression to activate
innate immunity18–20. Circulating exosomes expressing spike protein were detected in vaccinated participants21
and the S1 subunit, which is the subunit of the spike protein containing the receptor-binding domain (RBD),
responsible for binding to ACE2, was also d
etected22. Additionally, a recent report indicated that circulating free
spike antigen was present in adolescents and young adults with myocarditis after mRNA vaccination, although
extensive antibody profiling and T-cell responses were indistinguishable between patients with myocarditis
and healthy c ontrols23. Histopathological analyses of endomyocardial biopsies or autopsies from patients with
acute myocarditis following COVID-19 vaccination have revealed spike proteins and spike receptor binding
domain (S-RBD) in the cardiomyocytes of several p
atients24,25. These findings suggest that the spike protein or
its subunits/peptide fragments reach the heart, where they may directly change the gene expression and trigger
an innate immune response.
S-RBD is the major target and a common domain for various types of COVID-19 vaccines that interfere
with viral receptor b
inding6,26–28. Some studies have revealed that S-RBDs can cause inflammation. S-RBD promoted the activation and maturation of human dendritic cells with the activation of NF-kβ pathway29. S-RBD
also significantly aggravated lipopolysaccharide-induced acute lung injury in an in vivo mouse model through
NF-kβ pathway30,31. However, the effects of S-RBD on the innate immune response of cardiomyocytes have not
yet been elucidated.
This study aimed to investigate the direct effects of the SARS-CoV-2 S-RBD on human cardiomyocytes.
Here, we demonstrated the internalization of S-RBD into the induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) via ACE2 through the endolysosomal pathway. We assessed the direct effect of S-RBD on
the innate immune response in iPSC-CMs using quantitative PCR array analysis followed by single-cell RNA
sequence (scRNA-seq) analysis and found that S-RBD upregulated interferon (IFN)-responsive genes in mature
cardiomyocytes. ...
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