SARS-CoV-2 spike protein-mediated cardiomyocyte fusion may contribute to increased arrhythmic risk in COVID-19.

BACKGROUND: SARS-CoV-2-mediated COVID-19 may cause sudden cardiac death (SCD). Factors contributing to this increased risk of potentially fatal arrhythmias include thrombosis, exaggerated immune response, and treatment with QT-prolonging drugs. However, the intrinsic arrhythmic potential of direct SARS-CoV-2 infection of the heart remains unknown. OBJECTIVE: To assess the cellular and electrophysiological effects of direct SARS-CoV-2 infection of the heart using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). METHODS: hiPSC-CMs were transfected with recombinant SARS-CoV-2 spike protein (CoV-2 S) or CoV-2 S fused to a modified Emerald fluorescence protein (CoV-2 S-mEm). Cell morphology was visualized using immunofluorescence microscopy. Action potential duration (APD) and cellular arrhythmias were measured by whole cell patch-clamp. Calcium handling was assessed using the Fluo-4 Ca2+ indicator. RESULTS: Transfection of hiPSC-CMs with CoV-2 S-mEm produced multinucleated giant cells (syncytia) displaying increased cellular capacitance (75±7 pF, n = 10 vs. 26±3 pF, n = 10; P<0.0001) consistent with increased cell size. The APD90 was prolonged significantly from 419±26 ms (n = 10) in untransfected hiPSC-CMs to 590±67 ms (n = 10; P<0.05) in CoV-2 S-mEm-transfected hiPSC-CMs. CoV-2 S-induced syncytia displayed delayed afterdepolarizations, erratic beating frequency, and calcium handling abnormalities including calcium sparks, large "tsunami"-like waves, and increased calcium transient amplitude. After furin protease inhibitor treatment or mutating the CoV-2 S furin cleavage site, cell-cell fusion was no longer evident and Ca2+ handling returned to normal. CONCLUSION: The SARS-CoV-2 spike protein can directly perturb both the cardiomyocyte's repolarization reserve and intracellular calcium handling that may confer the intrinsic, mechanistic substrate for the increased risk of SCD observed during this COVID-19 pandemic.

Severe Acute Respiratory Syndrome Coronavirus 2, COVID-19, and the Renin-Angiotensin System: Pressing Needs and Best Research Practices.

The coronavirus disease 2019 (COVID-19) pandemic is associated with significant morbidity and mortality throughout the world, predominantly due to lung and cardiovascular injury. The virus responsible for COVID-19-severe acute respiratory syndrome coronavirus 2-gains entry into host cells via ACE2 (angiotensin-converting enzyme 2). ACE2 is a primary enzyme within the key counter-regulatory pathway of the renin-angiotensin system (RAS), which acts to oppose the actions of Ang (angiotensin) II by generating Ang-(1-7) to reduce inflammation and fibrosis and mitigate end organ damage. As COVID-19 spans multiple organ systems linked to the cardiovascular system, it is imperative to understand clearly how severe acute respiratory syndrome coronavirus 2 may affect the multifaceted RAS. In addition, recognition of the role of ACE2 and the RAS in COVID-19 has renewed interest in its role in the pathophysiology of cardiovascular disease in general. We provide researchers with a framework of best practices in basic and clinical research to interrogate the RAS using appropriate methodology, especially those who are relatively new to the field. This is crucial, as there are many limitations inherent in investigating the RAS in experimental models and in humans. We discuss sound methodological approaches to quantifying enzyme content and activity (ACE, ACE2), peptides (Ang II, Ang-[1-7]), and receptors (types 1 and 2 Ang II receptors, Mas receptor). Our goal is to ensure appropriate research methodology for investigations of the RAS in patients with severe acute respiratory syndrome coronavirus 2 and COVID-19 to ensure optimal rigor and reproducibility and appropriate interpretation of results from these investigations.

Development of a multiomics model for identification of predictive biomarkers for COVID-19 severity: a retrospective cohort study.

BACKGROUND: COVID-19 is a multi-system disorder with high variability in clinical outcomes among patients who are admitted to hospital. Although some cytokines such as interleukin (IL)-6 are believed to be associated with severity, there are no early biomarkers that can reliably predict patients who are more likely to have adverse outcomes. Thus, it is crucial to discover predictive markers of serious complications. METHODS: In this retrospective cohort study, we analysed samples from 455 participants with COVID-19 who had had a positive SARS-CoV-2 RT-PCR result between April 14, 2020, and Dec 1, 2020 and who had visited one of three Mayo Clinic sites in the USA (Minnesota, Arizona, or Florida) in the same period. These participants were assigned to three subgroups depending on disease severity as defined by the WHO ordinal scale of clinical improvement (outpatient, severe, or critical). Our control cohort comprised of 182 anonymised age-matched and sex-matched plasma samples that were available from the Mayo Clinic Biorepository and banked before the COVID-19 pandemic. We did a deep profiling of circulatory cytokines and other proteins, lipids, and metabolites from both cohorts. Most patient samples were collected before, or around the time of, hospital admission, representing ideal samples for predictive biomarker discovery. We used proximity extension assays to quantify cytokines and circulatory proteins and tandem mass spectrometry to measure lipids and metabolites. Biomarker discovery was done by applying an AutoGluon-tabular classifier to a multiomics dataset, producing a stacked ensemble of cutting-edge machine learning algorithms. Global proteomics and glycoproteomics on a subset of patient samples with matched pre-COVID-19 plasma samples was also done. FINDINGS: We quantified 1463 cytokines and circulatory proteins, along with 902 lipids and 1018 metabolites. By developing a machine-learning-based prediction model, a set of 102 biomarkers, which predicted severe and clinical COVID-19 outcomes better than the traditional set of cytokines, were discovered. These predictive biomarkers included several novel cytokines and other proteins, lipids, and metabolites. For example, altered amounts of C-type lectin domain family 6 member A (CLEC6A), ether phosphatidylethanolamine (P-18:1/18:1), and 2-hydroxydecanoate, as reported here, have not previously been associated with severity in COVID-19. Patient samples with matched pre-COVID-19 plasma samples showed similar trends in muti-omics signatures along with differences in glycoproteomics profile. INTERPRETATION: A multiomic molecular signature in the plasma of patients with COVID-19 before being admitted to hospital can be exploited to predict a more severe course of disease. Machine learning approaches can be applied to highly complex and multidimensional profiling data to reveal novel signatures of clinical use. The absence of validation in an independent cohort remains a major limitation of the study. FUNDING: Eric and Wendy Schmidt.

Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.