Introduction. Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) disease (COVID-19) is defined as a respiratory disease, but cardiac complications including myocarditis have been reported in almost 30% of cases. Nevertheless, the direct effect of SARS-CoV-2 infection on human cardiomyocytes is still unknown. The entry of SARS-CoV-2 in host cells is mediated by the interaction between the viral Spike (S) glycoprotein and the host angiotensin-converting enzyme 2 (ACE-2) receptor. A proteolytic cleavage by the host transmembrane serine protease II (TMPRSS2) or other ubiquitary proteases is also needed for S priming. Aims of the work. 1) Generate human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and prove their true cardiac identity; 2) establish that our hiPSC-CM express ACE-2 but not TMPRSS2 accordingly to literature; 3) assess if they are permissive to SARS-CoV-2 infection; 4) Use of hiPSC-CM to test antiviral and/or cardioprotective activity of drugs. Methods. We differentiated hiPSC into hiPSC-CMs, using a commercially-available kit. The obtained iPSC-CMs were studied after 20 days from differentiation induction and compared to a commercially-available embryonic stem cell derived-CM line (Cytiva). ACE-2 and TMPRSS2 mRNA levels were quantified by RT-qPCR. ACE-2 total protein content was evaluated by western blot, whereas the amount of soluble form was quantified by ELISA. A lung epithelial cancer cell line, which expresses high levels of ACE-2 and TMPRSS2 mRNAs and is permissive to SARS-CoV-2 infection, was used as positive control. Pulmonary cells, Cytiva and hiPSC-CMs were incubated with SARS-CoV-2 wild strain, isolated from a positive patient. After one hour, the virus was removed, and after 48 hours, cells were analyzed by light microscopy to check if they present a cytopathic effect (CPE). Then, the cells were fixed in paraformaldehyde 4% and viability was measured with anti troponin T (cTnT) immunostaining and in cell western (ICW) technique. Supernatants were collected and titrated to check for the presence of infectious virus in a plaque-forming assay on VERO E6 cells. The same experiments were repeated after a blinded pharmacologic treatment with three drugs (indicated as DRUG1, DRUG2 and DRUG3) at two concentrations (C1 and C2) for a week. Results/Discussion. Both iPSC-CMs and Cytiva display a proper cardiac phenotype, and express ACE-2 mRNA and protein, whereas TMPRSS2 is not expressed as expected. No cytopathic effect was observed in Pulmonary cells after 48 hrs of SARS-CoV-2 direct infection. On the contrary, hiPSC-CM and Cytiva viability was significantly reduced after viral infection, and we also observed a disorganization and fragmentation of TnT myofibrils. Viral titration revealed that SARS-CoV-2 replicated in hiPSC-CMs and Cytiva and was actively released in supernatants as in the control Pulmonary cells. DRUG1, DRUG2 and DRUG3 at both C1 and C2 did not affect viability of Pulmonary cells and iPSC-CMs in the absence of virus. After infection, we observed that DRUG2 at C2 significantly increases Pulmonary cell and iPSC-CM viability, and reduces supernatant viral titer. This finding suggests that DRUG2 may have a cardioprotective and antiviral activity, although further investigations are needed to determine the underlying mechanism of action. Conclusions. hiPSC-CMs express ACE-2 but not TMPRSS2, and are permissive to SARS-CoV-2 infection. These data indicate that iPSC-CMs are a suitable in vitro model to elucidate the underlying mechanisms of viral cardiac toxicity and to test novel antiviral drugs.
SARS-CoV-2 è il coronavirus responsabile della sindrome respiratoria acuta grave anche chiamata malattia da coronavirus 2019 (COVID-19). Quest’ultima colpisce in primis i polmoni, ma anche altri organi quali il cuore e il sistema gastrointestinale. Per quanto concerne il danno cardiaco, la miocardite è stata riscontrata in quasi il 30% dei casi con COVID-19. Nonostante queste evidenze cliniche, non è ancora certo che il virus possa infettare direttamente i cardiomiociti. L’entrata di SARS-CoV-2 nelle cellule ospite avviene mediante l’interazione tra il recettore Angiotensin-converting enzyme 2 (ACE2) e la proteina virale Spike (S). Inoltre, un taglio proteolitico da parte della serina proteasi II transmembrana (TMPRSS2) dell’ospite o di altri proteasi ubiquitarie è necessario per il S priming. Obiettivi. Generare cardiomiociti derivati da cellule staminali pluripotenti indotte umane (hiPSC-CM) e verificare la loro identità cardiaca; Stabilire se hiPSC-CM esprimono ACE2, ma non TMPRSS2 come riportato in letteratura. Valutare la loro permissività all’infezione da SARS-CoV-2; Usare le hiPSC-CM per testare l’attività antivirale e/o cardioprotettiva dei farmaci. Abbiamo differenziato hiPSC in hiPSC-CM, usando un kit commerciale. Gli hiPSC-CM ottenuti sono stati studiati dopo 20 giorni dall’inizio della differenziamento e paragonati ad una linea commerciale di cardiomiociti derivati da cellule staminali embrionali (Cytiva). I livelli di mRNA di ACE2 e TMPRSS2 sono stati quantificati tramite RT-qPCR. Il contenuto proteico di ACE2 è stato valutato tramite Western Blot, mentre la forma solubile è stata quantificata tramite ELISA. Una linea cellulare polmonare (CP) è stata usata come controllo positivo, perchè nota per esprimere ACE2 e TMPRSS2 ad alti livelli ed essere permissiva all’infezione da SARS-CoV-2. CP, Cytiva, hiPSC-CM sono stati incubati con il SARS-CoV-2, isolato da un paziente positivo. Dopo un’ora, il virus è stato rimosso e dopo 48 ore, le cellule sono analizzate con il microscopio ottico per valutare l’effetto citopatico. Poi le cellule sono state fissate in paraformaldeide 4% e la vitalità è stata misurata utilizzando l’anticorpo contro la Troponina T cardiaca (cTnT) sia per immunocitochimica che In Cell Western (ICW). Il surnatante è stato raccolto e titolato tramite un saggio con cellule VERO E6 per controllare la presenza del virus. Gli stessi esperimenti sono stati ripetuti anche dopo il trattamento farmacologico di una settimana con tre farmaci in cieco (indicati come DRUG1, DRUG2 e DRUG3) a due concentrazioni (C1 e C2). Risultati/Discussione. iPSC-CMs e Cytiva mostrano un fenotipo cardiaco ed esprimono mRNA e proteina di ACE-2, mentre TMPRSS2 non è espresso come atteso. Nessun effetto citopatico è stato osservato in CP dopo 48 ore dall’infezione diretta con SARS-CoV-2. Per quanto riguarda hiPSC-CM e Cytiva, la vitalità è stata significativamente ridotta dopo l’infezione virale ed una disorganizzazione e frammentazione delle miofibrille di cTnT è stata osservata. La titolazione virale ha mostrato che SARS-CoV-2 si è replicato in hiPSC-CM e Cytiva ed è stato rilasciato nel surnatante come nelle CP usate come controllo. DRUG1, DRUG2 e DRUG3 ad entrambe C1 e C2 non influenzano la vitalità nelle CP e nei iPSC-CM in assenza di virus. Dopo infezione DRUG2_C2 incrementa significativamente la vitalità delle cellule polmonari e iPSC-CM e riduce il titolo virale nel surnatante. Questi risultati suggeriscono che DRUG2 potrebbe avere un effetto cardioprotettivo ed un’azione antivirale, sebbene ulteriori indagini dovranno essere condotte. Conclusioni. hiPSC-CMs esprimono ACE2 ma non TMPRSS2 e sono permissive all’infezione di SARS-CoV-2. Questi dati indicano che hiPSC-CMs sono un buon modello in vitro per studiare i meccanismi alla base della cardiotossicità indotta dall’infezione di SARS-CoV-2 e per testare l’azione antivirale di nuovi farmaci.
Cardiomiociti derivati da cellule staminali pluripotenti indotte umane usate come modello in vitro per studiare il danno cardiaco mediato dall'infezione di SARS-CoV-2
DEGAETANO, VINCENZO
2019/2020
Abstract
Introduction. Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) disease (COVID-19) is defined as a respiratory disease, but cardiac complications including myocarditis have been reported in almost 30% of cases. Nevertheless, the direct effect of SARS-CoV-2 infection on human cardiomyocytes is still unknown. The entry of SARS-CoV-2 in host cells is mediated by the interaction between the viral Spike (S) glycoprotein and the host angiotensin-converting enzyme 2 (ACE-2) receptor. A proteolytic cleavage by the host transmembrane serine protease II (TMPRSS2) or other ubiquitary proteases is also needed for S priming. Aims of the work. 1) Generate human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and prove their true cardiac identity; 2) establish that our hiPSC-CM express ACE-2 but not TMPRSS2 accordingly to literature; 3) assess if they are permissive to SARS-CoV-2 infection; 4) Use of hiPSC-CM to test antiviral and/or cardioprotective activity of drugs. Methods. We differentiated hiPSC into hiPSC-CMs, using a commercially-available kit. The obtained iPSC-CMs were studied after 20 days from differentiation induction and compared to a commercially-available embryonic stem cell derived-CM line (Cytiva). ACE-2 and TMPRSS2 mRNA levels were quantified by RT-qPCR. ACE-2 total protein content was evaluated by western blot, whereas the amount of soluble form was quantified by ELISA. A lung epithelial cancer cell line, which expresses high levels of ACE-2 and TMPRSS2 mRNAs and is permissive to SARS-CoV-2 infection, was used as positive control. Pulmonary cells, Cytiva and hiPSC-CMs were incubated with SARS-CoV-2 wild strain, isolated from a positive patient. After one hour, the virus was removed, and after 48 hours, cells were analyzed by light microscopy to check if they present a cytopathic effect (CPE). Then, the cells were fixed in paraformaldehyde 4% and viability was measured with anti troponin T (cTnT) immunostaining and in cell western (ICW) technique. Supernatants were collected and titrated to check for the presence of infectious virus in a plaque-forming assay on VERO E6 cells. The same experiments were repeated after a blinded pharmacologic treatment with three drugs (indicated as DRUG1, DRUG2 and DRUG3) at two concentrations (C1 and C2) for a week. Results/Discussion. Both iPSC-CMs and Cytiva display a proper cardiac phenotype, and express ACE-2 mRNA and protein, whereas TMPRSS2 is not expressed as expected. No cytopathic effect was observed in Pulmonary cells after 48 hrs of SARS-CoV-2 direct infection. On the contrary, hiPSC-CM and Cytiva viability was significantly reduced after viral infection, and we also observed a disorganization and fragmentation of TnT myofibrils. Viral titration revealed that SARS-CoV-2 replicated in hiPSC-CMs and Cytiva and was actively released in supernatants as in the control Pulmonary cells. DRUG1, DRUG2 and DRUG3 at both C1 and C2 did not affect viability of Pulmonary cells and iPSC-CMs in the absence of virus. After infection, we observed that DRUG2 at C2 significantly increases Pulmonary cell and iPSC-CM viability, and reduces supernatant viral titer. This finding suggests that DRUG2 may have a cardioprotective and antiviral activity, although further investigations are needed to determine the underlying mechanism of action. Conclusions. hiPSC-CMs express ACE-2 but not TMPRSS2, and are permissive to SARS-CoV-2 infection. These data indicate that iPSC-CMs are a suitable in vitro model to elucidate the underlying mechanisms of viral cardiac toxicity and to test novel antiviral drugs.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/12446