E-viri
Recenzirano
-
Macrae, Robyn G; Williams, Thomas L; Colzani, Maria T; Bloor, Stuart; Zhan, Jun Ru; Nyimanu, Duuamene; Kuc, Rhoda E; Maguire, Janet J; Lehner, Paul J; Sinha, Sanjay; Davenport, Anthony P
Circulation (New York, N.Y.), 11/2021, Letnik: 144, Številka: Suppl_1Journal Article
Abstract only Introduction: Cardiovascular comorbidities are a major risk factor in patients infected with SARS-CoV-2. SARS-CoV-2 Spike protein binds to host cell surface ACE2 to gain entry. ACE2 is subsequently downregulated by internalisation. We hypothesise that an ACE2 knockdown system in beating human embryonic stem cell-derived cardiomyocytes (hESC-CMs) will recapitulate the downregulation of ACE2, and reduce the ability of a SARS-CoV-2 Spike protein pseudotyped virus to infect these clinically relevant cells. Method: ACE2 was knocked down (KD) with CRISPR/Cas9 in hESCs, before differentiating to hESC-CMs and sub-culturing in 96 well plates. Sanger sequencing confirmed amino acid changes. Catalytic ACE2 activity was measured by mass spectrometry in wild-type versus KD by conversion of apelin-13 to apelin-12 in hESC-CMs supernatant. ACE2 activity was also measured by fluorescent substrate assay. Pseudotyped virus infection was visualised by high content screening in ACE2 KD versus wild-type (n=4). All data are mean±SEM. Results: Sequencing revealed two substitutions at ACE2 222-223 , and three deletions at ACE2 224-226 . This reduced ACE2 catalytic activity by ~60-70% by apelin-12 accumulation using mass spectrometry (Fig 1a), and fluorescent assay. Furthermore, KD reduced infection of hESC-CMs by pseudotyped virus to 27.3±9.6% of the cell population versus 74.8±4.7% for wild-type (Fig 1b,c). This is consistent with our previous data, showing DX600 (peptidic inhibitor of ACE2 catalytic site) significantly reduced infection of wild-type hESC-CMs to 20.5±6.5%. Conclusions: In conclusion, we generated a functional ACE2 knockdown in a beating cardiomyocyte cell model. Both the catalytic activity and the ability to bind SARS-CoV-2 Spike protein of ACE2 reduced, importantly indicating ACE2 is rate limiting for infection. We aim to use this system to further explore the cardiovascular consequences of SARS-CoV-2 infection and subsequent downregulation of ACE2.
Vnos na polico
Trajna povezava
- URL:
Faktor vpliva
Dostop do baze podatkov JCR je dovoljen samo uporabnikom iz Slovenije. Vaš trenutni IP-naslov ni na seznamu dovoljenih za dostop, zato je potrebna avtentikacija z ustreznim računom AAI.
Leto | Faktor vpliva | Izdaja | Kategorija | Razvrstitev | ||||
---|---|---|---|---|---|---|---|---|
JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
Baze podatkov, v katerih je revija indeksirana
Ime baze podatkov | Področje | Leto |
---|
Povezave do osebnih bibliografij avtorjev | Povezave do podatkov o raziskovalcih v sistemu SICRIS |
---|
Vir: Osebne bibliografije
in: SICRIS
To gradivo vam je dostopno v celotnem besedilu. Če kljub temu želite naročiti gradivo, kliknite gumb Nadaljuj.