SARS-CoV-2,心肌细胞 z-bo 2020-12-18 380

　　COVID-19 is a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is defined by respiratory symptoms, but heart complications including viral myocarditis are also common. Although the ischemia and inflammation caused by COVID-19 can impair heart function, the direct impact of SARS-CoV-2 infection on human cardiomyocytes is unclear. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model to examine the mechanism of SARS-CoV-2-induced cardiomyocyte-specific infection. Microscopy and RNA sequencing indicate that SARS-CoV-2 can enter hiPSC-CMs through ACE2. SARS-CoV-2 virus replication and cytopathic effects can cause hiPSC-CM to apoptosis and stop beating after 72 hours of infection. SARS-CoV-2 infection can activate the innate immune response and antiviral clearance gene pathway, while inhibiting the metabolic pathway and inhibiting the expression of ACE2. These studies show that SARS-CoV-2 can infect hiPSC-CM in vitro, thereby establishing a model to clarify the mechanism of infection and a potential cardiac-specific antiviral drug screening platform.

　　 Introduction: SARS-CoV-2 is known to be a single-stranded enveloped RNA virus, which uses the ACE2 receptor to enter the host lung tissue and then rapidly replicate the virus. The clinical symptoms are mainly pulmonary symptoms, including cough, shortness of breath, pneumonia and acute respiratory distress syndrome. However, there is increasing evidence that SARS-CoV-2 infection may cause cardiac complications, including elevated cardiac stress biomarkers, arrhythmia, and heart failure. A recent study showed that troponin levels in some patients with COVID-19 were significantly increased, indicating that heart damage, especially heart damage, is associated with an increased risk of death. However, the cause of COVID-19 myocardial injury is still unclear. Heart damage may be mediated by ischemia, and inflammation and hemodynamic effects are thought to be the cause of atherosclerotic plaque rupture or imbalance of oxygen supply and demand leading to ischemia. In addition, myocardial tissue expresses the ACE2 receptor, which further indicates the feasibility of SARS-CoV-2 to be directly internalized in myocardial cells. ACE2 is also up-regulated in cardiomyocytes in dilated and hypertrophic cardiomyopathy. It is important to note that the related SARS-CoV virus is located in the myocardium, and recent studies have shown that there is 79% sequence conservation in the viral genomes of SARS-CoV and SARS-CoV-2. Structurally, both viruses use the ACE2 receptor to enter cells and bind with affinity similar to ACE2. In view of these similarities with SARS-CoV, it seems reasonable that SARS-CoV-2 can also use ACE2 to enter adult cardiomyocytes. The preliminary COVID-19 clinical case report has increased the suspicion of direct myocardial SARS-CoV-2 infection and fulminant myocarditis-mediated heart damage. A recent multi-organ autopsy study of COVID-19 patients detected SARS-CoV-2 viral RNA in the heart by PCR. However, the gold standard for confirming the localization of SARS-CoV-2 in cardiomyocytes is endocardial biopsy (EMB), which is an invasive procedure, and additional safety must be taken to perform EMB in the case of COVID-19. Precaution. Therefore, there are only limited clinical data to prove that SARS-CoV-2 is localized in adult cardiomyocytes. In order to further understand the cardiology of COVID-19, it is important to determine whether SARS-CoV-2 can directly infect isolated human cardiomyocytes. Elucidating the pathogenic mechanism of COVID-19 in vitro heart injury can ultimately guide treatment strategies. If the underlying mechanism of heart damage is direct myocardial viral infection, antiviral drugs may reduce heart complications.

　　It is difficult to obtain and maintain primary human cardiomyocytes for research. An improved method of converting human induced pluripotent stem cells (hiPSC) into a variety of somatic cell lines enables the mass production of patient-specific cells in vitro, including hiPSC-derived cardiomyocytes (hiPSC-CM). hiPSC-CMs express related proteins found in adult CMs, can shrink spontaneously, can be made within a few weeks using a defined differentiation protocol, and can be genetically customized using genome editing. HiPSC CMs express ACE2, and its expression increases after 90 days of differentiation. hiPSC-CM also responds to drugs (such as norepinephrine) and can control the pulsation rate through electrical stimulation. Since hiPSC-CMs can be purified and transplanted for downstream applications, research groups in academia and industry are now using these cells for cardiovascular disease modeling and high-throughput drug screening analysis. HiPSC-CMs can reproduce the cellular phenotype of cardiovascular diseases, including various forms of cardiomyopathy and drug-induced cardiotoxicity. It is worth noting that hiPSC-CMs also show promise as an in vitro model for studying the mechanism of viral myocarditis directly infecting cardiomyocytes.

　　 Previous studies have shown that CVB3 is one of the main pathogens of viral myocarditis, which can rapidly infect and proliferate in hiPSC-CMs. Like SARS-CoV-2, CVB3 is a sense single-stranded RNA virus, although unlike SARS-CoV-2, it does not have a viral envelope. hiPSC-CMs produce coxsackie and adenovirus receptor proteins, which are required for CVB3 infection. Within a few hours after infection with CVB3, harmful virus-induced cytopathic changes were observed in hiPSC CMs, which manifested as cell death and irregular contraction. Importantly, the study also established hiPSC-CMs as a heart-specific antiviral drug screening platform, and proved that drugs such as interferon beta and ribavirin can hinder the proliferation of CVB3 in vitro. "The aforementioned basic viral myocarditis study has been extended to evaluate the effect of SARS-CoV-2 on hiPSC-CM. It shows that SARSCoV-2 is susceptible to infecting hiPSC-CMs, leading to functional changes, transcription changes and cytopathic effects. These cell phenotypes occur without the influence of systemic inflammation and hemodynamics, establishing an in vitro cardiac platform to study SARS-CoV-2 infection.

　　Result: Human induced pluripotent stem cells can differentiate into cardiomyocytes:

　　 Cedars-Sinai Medical Center iPSC Core produced a hiPSC control line from peripheral blood mononuclear cells, showing that it is pluripotent. Using small molecule regulators of Wnt signaling, the established monolayer differentiation protocol was used to differentiate hiPSC into hiPSC-CMs. As mentioned earlier, differentiated hiPSC-CMs are metabolically purified by removing glucose in the cell. The purified hiPSC-CMs express the standard cardiac sarcomere markers cardiac troponin T (cTnT) and α-actin.

　　SARS-CoV-2 can directly infect purified hiPSC-CM:

　　 The purified hiPSC-CM was re-seeded into a 96-well plate with 100,000 cells per well, and the contractility was restored before SARS-CoV-2 infection. SARS-CoV-2 was obtained from the Biodefense and Emerging Infection (BEI) resources of the National Institute of Allergy and Infectious Diseases (NIAID) and was titrated on Vero-E6 cells. Unless otherwise specified, the dose response and time course were performed on hiPSC-CM, and the multiplicity of infection (MOI) for all experiments was selected as 0.1. In all experiments, SARS-CoV-2 infected hiPSC-CMs for up to 72 hours, unless otherwise specified, the virus-free mock treatment was used as the control condition. Plaque formation analysis of the supernatant collected after hiPSC-CM infection confirmed that SARS-CoV-2 infection was active. The infected hiPSC-CM stained positively for the spike protein, which indicates that SARS-CoV-2 can establish active infection in hiPSC-CM. As mentioned earlier, pretreatment of infected hiPSC-CMs with ACE2 antibody can significantly reduce viral protein expression and plaque formation, indicating that ACE2 is essential for SARS-CoV-2 internalization.

　　 SARS-CoV-2 infection with hiPSC-CMs can cause cell apoptosis and pulsation arrest: In order to determine whether SARS-CoV-2 has a cytopathic effect on hiPSC-CMs, the apoptosis markers of simulated and infected hiPSC-CMs cut caspase-3 and double-stranded RNA (dsRNA) that are unique to positive sense RNA virus infection. The product is dyed. dsRNA and spike protein staining are two independent detection methods used to show the uptake and genome replication of SARSCoV-2 virus in hiPSC-CMs. A certain proportion of infected cells were positive for dsRNA and caspase-3 lysis, indicating that hiPSC-CM is undergoing virus-induced apoptosis. Pretreatment of infected hiPSC-CMs with ACE2 antibody can significantly reduce the expression of caspase-3 and inhibit cell apoptosis. It is worth noting that the SARS-CoV-2 spike protein is located in the pernuclear region of hiPSC-CMs, which is consistent with the previous results of coronavirus infection in non-cardiomyocytes and coxsackie virus infection on hiPSC-CMs. The dsRNA intermediate product shows cytoplasmic localization. Quantification of stained cells showed the percentage of total cells positive for dsRNA, spike protein, and lysed caspase-3. The dsRNA intermediate product appeared as a cytoplasmic localized infection, while the mock pore continued to shrink. In summary, these results indicate that hiPSC CMs are susceptible to ACE2-mediated SARS-CoV-2 infection and downstream harmful cytopathic effects. SARS-CoV-2 may select organelles for viral protein translation, which is different in hiPSC CMs. Duplication of the pernuclear location, SARS-CoV-2 infection can significantly reduce the contractile function of hiPSC-CMs.

　　 Transcription analysis of hiPSC-CM infected with SARS-CoV-2:

　　 After 72 hours of infection of hiPSC-CMs with 0.1 MOI SARS-CoV-2, transcriptomics analysis was performed by RNA sequencing. After mapping the genome reads of infected hipscms, more than 50% of the map reads came from the SARS-CoV-2 genome, further indicating that there is active SARS-CoV-2 virus genome replication in the infected hiPSC-CM. The most significant expression change is CXCL2, which encodes an immune cytokine that is known to be transcriptionally upregulated during SARS-CoV infection. Similarly, immunomodulators (such as interleukins) and antiviral response pathway genes (such as OAS3) in infected samples were also significantly up-regulated. In infected samples, the expression of cardiomyocyte markers TNNT2 and TNNC1 and the expression of mitochondrial genes related to oxidative phosphorylation (such as CKMT2) were significantly reduced, which encodes creatine kinase. It is worth noting that the expression of ACE2 was significantly down-regulated in hiPSC-CMs infected with SARS-CoV-2, which is consistent with previous reports of detection of SARS-CoV myocardial infection. Gene pathway analysis confirmed that transcription pathways related to mitochondrial function, oxidative phosphorylation and heart function were down-regulated, while transcriptional up-regulated pathways included responses to organics, immune system processes and apoptosis. These results indicate that SARS-CoV-2 infection induced significant transcriptional changes in gene pathways related to cellular metabolism and immune response in hiPSC-CMs.

　　 Discussion: The results of this study confirmed that human iPSC-derived cardiomyocytes are susceptible to direct infection of ACE2-mediated SARS-CoV-2, and the virus may induce harmful cytopathic changes in these cells. When MOI is 0.1, hiPSC-CM will have cytopathic effect 72 hours after infection. It is speculated that the virus enters the cell through the ACE2 receptor, which is expressed by hiPSC-CM in this study. ACE2 receptor antibodies can significantly inhibit viral protein expression, replication and cell death. Despite the use of ACE2 antibody, cells are still vulnerable to infection, which suggests a potential alternative mechanism for SARS-CoV-2 internalization. Immunostaining of the two components of SARS-CoV-2, namely the unique dsRNA intermediate product and the "spinous process" capsid protein responsible for virus entry and virus particle assembly, showed that this new type of coronavirus can enter hipscms to release its RNA and hijack the translation mechanism of the host hiPSC-CM to produce new viral components. It is speculated that the increase in viral load in hiPSC-CM activates the apoptotic event. It is manifested in the change of cell morphology, the increase of lysed caspase-3 and the activation of cell death genes. The contractile force of hiPSC-CM undergoes a functional change, which makes the cell stop beating. In hiPSC CMs infected with CVB3, similar calcium treatment and significant changes in contractility were also observed. In addition, SARS-CoV-2 induced extensive transcriptional changes in infected hiPSC-CM.

　　 In response to COVID-19, people have proposed a variety of antiviral methods, from recombinant small molecules, such as nucleoside analogs or viral polymerase inhibitors, to new antibodies and antisense oligonucleotides, and are currently undergoing in vitro and clinical trials. HiPSC-CMs can be used as cardiac-specific helper cells for in vitro preclinical efficacy studies of any drug intended to inhibit the proliferation of SARS-CoV-2. Given that some existing COVID-19 drug treatments exhibit these targeted cardiotoxicities, they can also be used in preclinical development to check for arrhythmias and QT interval prolongation caused by antiviral compounds.