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Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated., Williams et al. confirm that human embryonic stem cell-derived cardiomyocytes express the protein machinery critical for SARS-CoV-2 infection and are susceptible to SARS-CoV-2 spike protein pseudotyped virus infection. They further use this platform as a screen to identify inhibitors of SARS-CoV-2 infection, reporting benztropine (targeting B0AT1/ACE2 complex) and DX600 (targeting ACE2) as potential inhibitors.

Introduction Apelin receptor activation promotes beneficial vasodilation and positive inotropy1 and is critical for early cardiac development.2 In cardiovascular disease states, including heart failure, expression of apelin receptor endogenous peptide ligands, apelin and Elabela, are downregulated.1 We aim to use human embryonic stem cell (hESC)-derived cardiomyocytes (hESC-CMs) as a model to interrogate the role of the apelin receptor in the cardiovascular system and the potential of targeting the receptor therapeutically. Here, we have generated a system to inducibly knockdown the apelin receptor in hESC-CMs to examine the effects on differentiation of hESC to cardiomyocyte, and the effects on function of the resulting hESC-CMs. Methods A short hairpin RNA based apelin receptor (shAPLNR) inducible knockdown system was generated in pluripotent hESCs, utilising the sOPTiKD system as described previously,3 before differentiating to hESC-CMs. Inclusion of tetracycline in the culture medium was used to induce knockdown, with knockdown efficiency determined by qRT-PCR and radioligand binding. RNA-sequencing analysis and a panel of phenotypic assays were performed to examine effects of apelin receptor knockdown on cardiomyocyte differentiation and function. Results We have previously demonstrated that hESC-derived cardiomyocytes express the apelinergic system at a similar level to adult cardiomyocytes (Bmax hESC-CMs 21 fmol/mg versus adult 14 fmol/mg). Apelin receptor expression was significantly reduced in hESCs and hESC-CMs by tetracycline inclusion at both RNA and protein level (84.2% ± 4.3 and 82.8% ± 3.9 compared to control in hESCs and hESC-CMs, respectively). Interestingly, efficiency of differentiation to cardiomyocyte was reduced compared to control cells (77.8% ± 5.5 cardiomyocyte control vs 22.2% ± 7.4 cardiomyocyte knockdown). RNA-sequencing analysis revealed 272 differentially expressed genes involved in pathways related to electrophysiological signalling, adhesion and the cytoskeleton. Discussion We have successfully shown knockdown of apelin receptor expression in hESC-CMs. To our knowledge, this is the first use of this system to knockdown expression of a GPCR. This system allows detailed characterisation of apelin receptor activation in cardiovascular development and disease pathogenesis, providing an innovative approach to develop strategies to target the apelin receptor therapeutically in cardiovascular disease, where novel treatments are urgently needed. Conflict of Interest None References Yang et al. Circulation 2017;135:1160–1173. Chng et al. Developmental Cell 2013;27(6):672–680. Bertero et al. Development 2016;143:4405–4418.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection.

Introduction The apelin receptor is expressed throughout the cardiovascular system, including in cardiomyocytes. Receptor activation by either of its endogenous peptide ligands, apelin or Elabela, has a positive inotropic effect and promotes vasodilatation. Human embryonic stem cell (hESC)-derived cells have the potential for use in translational research to investigate cellular signalling, disease pathogenesis and potential novel treatments. Our aim was to determine if hESC-derived cardiomyocytes express apelin receptor protein and to quantify receptor density to ascertain if this cell type can be used as a phenotypic model for human diseases associated with apelin receptor mutations. Methods H9 hESCs were cultured to induce differentiation to beating cardiomyocytes. Saturation radioligand binding experiments were performed using [Glp65,Nle75,Tyr77][125I]apelin-13 and [Pyr1]apelin-13 to define non-specific binding. Bound radioactivity was counted and data analysed using iterative curve fitting programs to obtain values of receptor affinity (KD) and density (BMAX). Immunocytochemistry was carried out using anti-apelin receptor or anti-cardiac cell marker antibodies. Results Previous work demonstrated expression of the apelin receptor in hESC-derived cardiomyocytes at the gene level by qRT-PCR at similar levels to adult cells (figure 1A). Here, radioligand binding studies have confirmed receptor protein expression in beating hESC-derived cardiomyocytes. Binding was saturable and [125I]apelin-13 bound with sub-nanomolar affinity (0.12 nM) and receptor density found to be 21 fmol/mg (figure 1B), comparable to that found in human adult heart. Hill slope was close to 1 consistent with a single binding site for apelin in these cells. Furthermore, beating hESC-derived cardiomyocytes stained positive for apelin receptor, in addition to the standard cardiac markers including cardiac troponin T (figure 2). Conclusion These data importantly confirm that hESC-derived cardiomyocytes express apelin receptor protein at similar levels to adult human heart. Apelin receptor mutations have been identified in the 100,000 Genomes Bridge Project that are associated with rare diseases, including pulmonary arterial hypertension. Our ongoing experiments aim to pharmacologically characterise the apelinergic signalling pathway in the beating hESC-derived cardiomyocytes and we propose to generate hESC-derived phenotypic models by introducing selected apelin receptor mutations via CRISPR/Cas-9 gene editing. Conflict of interest None