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Angiotensin-converting enzyme 2 (ace2) is rate-limiting for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) viral infection in beating stem cell-derived cardiomyocytes
Circulation ; 144(SUPPL 1), 2021.
Article in English | EMBASE | ID: covidwho-1634978
ABSTRACT

Introduction:

Cardiovascular comorbidities are a major risk factor in patients infected with SARSCoV-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 , and three deletions at ACE2 . 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.
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Full text: Available Collection: Databases of international organizations Database: EMBASE Language: English Journal: Circulation Year: 2021 Document Type: Article

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Full text: Available Collection: Databases of international organizations Database: EMBASE Language: English Journal: Circulation Year: 2021 Document Type: Article