key: cord-0429252-pwlle828 authors: Robinson, Emma L.; Alkass, Kanar; Bergmann, Olaf; Maguire, Janet J.; Roderick, H. Llewelyn; Davenport, Anthony P. title: Genes Encoding ACE2, TMPRSS2 and Related Proteins Mediating SARS-CoV-2 Viral Entry are Upregulated with Age in Human Cardiomyocytes date: 2020-07-07 journal: bioRxiv DOI: 10.1101/2020.07.07.191429 sha: 575e1e9675e72cb40839507570e390267c5cb98d doc_id: 429252 cord_uid: pwlle828 Age is an independent risk factor for adverse outcome in patients following COVID-19 infection. We hypothesised that differential expression of genes encoding proteins proposed to be required for entry of SARS-Cov-2 in aged compared to younger cardiomyocytes might contribute to the susceptibility of older individuals to COVID-19-associated cardiovascular complications. We generated strand-specific RNA-sequencing libraries from RNA isolated from flow-sorted cardiomyocyte nuclei from left ventricular tissue. RNASeq data were compared between five young (19-25yr) and five older (63-78yr) Caucasian males who had not been on medication or exhibited evidence of cardiovascular disease post-mortem. Expression of relevant genes encoding ACE2, TMPRSS2, TMPRS11D, TMPRS11E, FURIN, CTSL, CTSB and B0AT1/SLC6A19 were upregulated in aged cardiomyocytes and the combined relative cardiomyocyte expression of these genes correlated positively with age. Genes encoding proteins in the RAAS and interferon/interleukin pathways were also upregulated such as ACE, AGTR1, MAS1 and IL6R. Our results highlight SARS-CoV-2 related genes that have higher expression in aged compared with young adult cardiomyocytes. These data may inform studies using selective enzyme inhibitors/antagonists, available as experimental compounds or clinically approved drugs e.g. remdesivir that has recently been rapidly accepted for compassionate use, to further understand the contribution of these pathways in human cardiomyocytes to disease outcome in COVID-19 patients. 3 Age (>70 years), case fatality rate (CFR, 10 .2%) and coexisting conditions, particularly cardiovascular disease (CFR, 10 .5%) and hypertension (CFR,6.0%), are independent predictors of adverse outcome for 45,000 COVID-19 patients in China 1 . A consensus has emerged that SARS-CoV-2 uses the same 'receptor' as SARS-CoV, the angiotensin converting enzyme 2 (ACE2), for initial binding to the host cell. This must be co-expressed with the serine protease TMPRSS2, that primes the spike protein S1 for endocytosis-mediated internalization of virus, employing the S2 domain for fusion to the host membrane ( Figure [1A] ) [2] [3] [4] CoV-2 is a spike protein second site (S2'), proposed to be cleaved by the proteinase furin 2 . Once inside the cell cysteine proteases, cathepsin L and B, are thought critical for endosomal processing in certain cells 3, 4 . In the cardiovascular system, ACE2 protein 5 and mRNA 6 are present in cardiomyocytes. ACE2 normally functions as a carboxypeptidase cleaving single Cterminal amino acids thus hydrolysing Pro-Phe in Ang-II to Ang1-7, [Pyr 1 ]-apelin-13 to [Pyr 1 ]-apelin(1-12) and des-Arg 9 -bradykinin to inactive bradykinin(1-8) 4 . Internalization of ACE2 by virus potentially reduces the beneficial counter regulatory function of these peptide products to the RAAS pathway 2, 4 . Conversely, the serine protease ADAM17 cleaves ACE2 to release its ectodomain and this is stimulated by Ang-II and, potentially, apelin acting via their respective G-protein coupled receptors 7 . Shed ACE2 binds SARS-CoV-2, a complex predicted not to internalize, and therefore circulating ACE2 could be exploited as a beneficial viral decoy substrate. Intriguingly, ACE2 is highly expressed in the GI tract where it is associated with B 0 AT1 (SLC6A19) that actively transports most neutral amino acids across the apical membrane of epithelial cells 4, 8 . It is not yet known if B 0 AT1 and ACE2 are coexpressed in cardiomyocytes and represent an important mechanism of viral entry, but they can form a heterodimer, with the ACE2 capable of binding the spike protein S1 8 . Interleukin 6 (IL-6), normally transiently produced, is elevated in serum and positively correlated with disease severity in COVID-19 patients 9 . We hypothesised that differential expression of genes encoding proteins in these pathways in aged cardiomyocytes could explain why the myocardium of older patients might be particularly vulnerable to the virus, manifesting as cardiovascular complications such as myocarditis. To selectively analyse cardiomyocyte gene expression, we generated strand-specific RNA-sequencing libraries from RNA 4 isolated from flow-sorted cardiomyocyte nuclei from left ventricular tissue 10 were expressed in cardiomyocytes, no pattern emerged with age. 5 Our results highlight SARS-CoV-2 related genes that have higher expression in aged compared with young adult cardiomyocytes. Our objective is to foster confirmation (or rebuttal) by studies on relevant protein levels in cardiomyocytes and using selective enzyme inhibitors/antagonists to dissect these pathways; available as experimental compounds or clinically approved drugs e.g. remdesivir has recently been rapidly accepted for compassionate use References are limited to ten but see recent review 5 for a comprehensive list of references supporting the concepts outlined in this letter. [A] Schematic diagram of the key proteins predicted from RNASeq data to be expressed by human cardiomyocytes. We propose SARS-CoV-2 binds initially to ACE2 (with the ACE2/B 0 AT1 complex as a potential second entry site). TMPRSS2 priming of the spike protein S1 together with further protease activation by [E] Schematic diagram of the genes encoding GPCRs that are expressed and higher in expression in aged human cardiomyocytes compared with young. Activation of the renin-angiotensin system, with overproductuon of Ang-II, contributes to acute respiratory distress syndrome following infection by coronoviruses. The Ang-II synthetic enzyme ACE and the ANG-II cognate receptor gene AGTR1 increased with age, together with B1 receptor gene, BDKBR1. This bradykinin receptoris selectively activated by des-Arg 9 -bradykinin (that would normally be inactiveated by ACE2), representing a second deleterious pathway. Both pathways could be blocked with clinically approved drugs (ACE inhibitors, AT1 receptor antagonists and the B2 receptor antagonist Icatibant) for secondary treatment in pateints with COVID-19. Internalization of ACE2 by the virus is predicted to increase Ang II levels but reduce those of Ang1-7 but the gene encoding the proposed Ang1-7 receptor, MAS, was still detected in aged myocytes, as was the apelin receptor. The beneficial potent positive inotropic action of apelin in the heart as well as its anti-thrombotic and anti-diabetic properties, suggests its receptor is a promising therapeutic target for administered apelin and this strategy is currently under clinical investigation. 10 Figure 1 Preventing a covid-19 pandemic: ACE inhibitors as a potential risk factor for fatal Covid-19 The Science Underlying COVID-19: Implications for the Cardiovascular System SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor A rational roadmap for SARS-CoV-2/COVID-19 pharmacotherapeutic research and development. IUPHAR Review 29 The protein expression profile of ACE2 in human tissues bioRxiv Myocyte Specific Upregulation of ACE2 in Cardiovascular Disease: Implications for SARS-CoV-2 mediated myocarditis Clinical Relevance and Role of Neuronal AT1 Receptors in ADAM17-Mediated ACE2 Shedding in Neurogenic Hypertension Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 Tocilizumab for the treatment of severe COVID-19 pneumonia with hyperinflammatory 7 syndrome and acute respiratory failure: A single center study of 100 patients in The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy Heart samples were obtained from the KI Donatum, Karolinska Institutet, Stockholm, Sweden with ethical approval. Quality control and sequencing was carried out at The Babraham Institute Sequencing Facility, Cambridge (UK).