ACE2 (Angiotensin-Converting Enzyme 2) and TMPRSS2 (Transmembrane Serine Protease 2) Expression and Localization of SARS-CoV-2 Infection in the Human Heart.

Abstract

HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 41, No. 1ACE2 (Angiotensin-Converting Enzyme 2) and TMPRSS2 (Transmembrane Serine Protease 2) Expression and Localization of SARS-CoV-2 Infection in the Human Heart Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBACE2 (Angiotensin-Converting Enzyme 2) and TMPRSS2 (Transmembrane Serine Protease 2) Expression and Localization of SARS-CoV-2 Infection in the Human Heart Atsushi Sakamoto, Rika Kawakami, Kenji Kawai, Andrea Gianatti, Dario Pellegrini, Robert Kutys, Liang Guo, Masayuki Mori, Anne Cornelissen, Yu Sato, Antonio Bellasi, Lara Faggi, Charles Hong, Maria Romero, Giulio Guagliumi, Renu Virmani and Aloke V. Finn Atsushi SakamotoAtsushi Sakamoto CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). *These authors contributed equally to this article. Search for more papers by this author , Rika KawakamiRika Kawakami CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). *These authors contributed equally to this article. Search for more papers by this author , Kenji KawaiKenji Kawai CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). *These authors contributed equally to this article. Search for more papers by this author , Andrea GianattiAndrea Gianatti Ospedale Papa Giovanni XXIII Hospital, Bergamo, Italy (A.G., D.P., A.B., L.F., G.G.). Search for more papers by this author , Dario PellegriniDario Pellegrini https://orcid.org/0000-0002-5545-585X Ospedale Papa Giovanni XXIII Hospital, Bergamo, Italy (A.G., D.P., A.B., L.F., G.G.). Search for more papers by this author , Robert KutysRobert Kutys CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). Search for more papers by this author , Liang GuoLiang Guo CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). Search for more papers by this author , Masayuki MoriMasayuki Mori CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). Search for more papers by this author , Anne CornelissenAnne Cornelissen https://orcid.org/0000-0002-5452-4657 CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). Search for more papers by this author , Yu SatoYu Sato CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). Search for more papers by this author , Antonio BellasiAntonio Bellasi https://orcid.org/0000-0001-7830-1645 Ospedale Papa Giovanni XXIII Hospital, Bergamo, Italy (A.G., D.P., A.B., L.F., G.G.). Search for more papers by this author , Lara FaggiLara Faggi Ospedale Papa Giovanni XXIII Hospital, Bergamo, Italy (A.G., D.P., A.B., L.F., G.G.). Search for more papers by this author , Charles HongCharles Hong https://orcid.org/0000-0002-0424-8252 University of Maryland, Baltimore (C.H., A.V.F.). Search for more papers by this author , Maria RomeroMaria Romero CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). Search for more papers by this author , Giulio GuagliumiGiulio Guagliumi Ospedale Papa Giovanni XXIII Hospital, Bergamo, Italy (A.G., D.P., A.B., L.F., G.G.). Search for more papers by this author , Renu VirmaniRenu Virmani https://orcid.org/0000-0003-1879-0015 CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). Search for more papers by this author and Aloke V. FinnAloke V. Finn Correspondence to: Aloke V. Finn, MD, CVPath Institute, Inc, 19 Firstfield Rd, Gaithersburg, MD 20878. Email E-mail Address: [email protected] https://orcid.org/0000-0003-0359-0184 CVPath Institute, Inc, Gaithersburg, MD (A.S., R. Kawakami, K.K., R. Kutys, L.G., M.M., A.C., Y.S., M.R., R.V., A.V.F.). University of Maryland, Baltimore (C.H., A.V.F.). Search for more papers by this author Originally published22 Oct 2020https://doi.org/10.1161/ATVBAHA.120.315229Arteriosclerosis, Thrombosis, and Vascular Biology. 2021;41:542–544Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: October 22, 2020: Ahead of Print At least 20% of patients hospitalized with coronavirus disease 2019 (COVID-19) have evidence of cardiac injury and its presence increases mortality.1 ACE 2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) facilitate SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) viral infection. Although ACE2 is expressed in the respiratory epithelium, cardiac tropism for SARS-CoV-2 remains uncertain. Several reports have assessed ACE2 and TMPRSS2 expression by RNA sequencing of normal and diseased hearts2,3 and reported that ACE2 expression was the highest in pericytes, but it was also appreciable in other cell types including cardiomyocytes, suggesting the heart may be a target for SARS-CoV-2 infection.To understand which cells in the heart are the most likely targets for infection, immunostaining for ACE2 and TMPRSS2, real-time polymerase chain reaction, and in-situ hybridization (ISH) of hearts from 15 COVID-19 victims from Bergamo, Italy was conducted. Control normal hearts, dilated cardiomyopathy, and viral myocarditis hearts (non-COVID-19) were selected from CVPath registry. The study was approved by the ethical committee at Ospedale Papa Giovanni XXIII and CVPath. Anti-ACE2 (R&D), anti-TMPRSS2 (SantaCruz), anti-CD31, anti-CD146, and anti-Connexin-43 (all Abcam) antibodies were applied. The specificity of anti-ACE2 antibody was confirmed by preincubation with recombinant ACE2 (Novus). RNA was extracted from the lung and hearts of COVID-19 victims. SARS-CoV-2 genes were amplified by TaqMan with 2019-nCoV CDC EUA kit (IDT). Virus-copy number was estimated using 2019-nCoV plasmid control (IDT). A SARS-CoV-2 RNA scope ISH (ACDBio) was visualized by confocal microscopy (Zeiss).ACE2 was detectable at low levels in normal hearts, mainly localized to myocytes. ACE2 was increased in dilated cardiomyopathy and was lower in myocarditis (non-COVID-19) and the lowest in COVID-19 hearts (Figure [A] and [B]). TMPRSS2 followed a similar expression pattern and was localized to myocytes but was not different across the different these groups (Figure [A] and [C]). ACE2 in myocytes was found mainly at plasma membranes (Figure [D]), both in normal and dilated cardiomyopathy hearts. TMPRSS2 predominantly localized to the nucleus and cytoplasm (Figure [D]). We also examined the expression of ACE2 and TMPRSS2 in pericytes and endothelial cells (intramyocardial vessels and coronary arteries). ACE2 was barely appreciable in the endothelium and pericytes of intramyocardial microvessels, but TMPRSS2 could be found in the latter (Figure [E] and [F]). ACE2 and TMPRSS2 were readily detectable in the endothelium of coronary arteries (Figure [G]). Figure [H] and [I] show the ACE2 and TMPRSS2 quantitative expression in myocytes, pericytes, microvascular, and coronary artery endothelial cells in normal and dilated cardiomyopathy hearts. A recent sc-RNA seq-based analysis showed ACE2 expression in the adult human heart was higher than lung,3 although the major susceptible organ for SARS-CoV-2 is the lung. Moreover, minimal gene expression of TMPRSS2 in the adult heart and coronary artery were also shown.2,3 Our observations about ACE2 and TMPRSS2 protein expression in the heart and coronary artery are inconsistent with these RNA sequencing-based reports.We next investigated whether SARS-CoV-2 could be located in the heart by real-time polymerase chain reaction in 15 COVID-19 hearts. The median age was 72 years (interquartile range, 58–80) and 73.3% were male. Five cases had a history of cardiovascular disease. Seven had evidence of cardiac injury (elevated cardiac enzymes or acute abnormal findings by ECG or echocardiogram) during hospitalization. Causes of death included respiratory failure (53.3%), acute myocardial infarction (13.3%), pulmonary embolism (20.0%), and septic shock (13.3%). Histological examination revealed 2 acute myocardial infarction cases and 7 with severe stenosis in at least one coronary artery. Eleven had the evidence of myocardial hypertrophy, but none of the 15 cases showed pathological findings consistent with myocarditis according to established criteria (T-lymphocytes and macrophages [>14 cells/mm2] with presence of CD3 positive T-lymphocytes [>7 mm2] around an area of myocyte necrosis [nonischemic], easily visualized at low-power magnification). Although SARS-CoV-2 could be found in the lung in most of the 15 cases (93.3%), the virus was not found in any chamber of the heart except for one case detected in the left atrium (6.7%; Figure [J]). The number of viral copies/tissue weight was markedly greater in the lung versus the left atrium (lung, 177.7 [28.4–2848.0] versus heart, 15.6; Figure [K]). ISH and indirect immunofluorescence revealed that SARS-CoV-2 could be found within myocytes in the left atrium (Figure [L] through [N]). By ISH, the number of viral-infected cells/mm2 field was lower in the heart than in infected lung (Figure [O] through [Q]; lung, 0.83 [0.75–10.3] versus heart, 0.16; Figure [R]).Download figureDownload PowerPointFigure. ACE2 (angiotensin-converting enzyme 2)/TMPRSS2 (transmembrane serine protease 2) expression and localization of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in human hearts.A, H&E and immunofluorescent images of ACE2 (red) and TMPRSS2 (purple) in normal hearts, dilated cardiomyopathy (DCM), non-coronavirus disease 2019 (COVID-19) viral myocarditis, and COVID-19 autopsy hearts. B and C, Percentage area of ACE2 (B) and TMPRSS2 (C) expression among the groups (n=3 in each). Different fields of interest (0.5 mm2) were randomly collected from endo-, mid-, and epicardial area of left ventricle in each case. Positive areas were analyzed by Image J (National Institutes of Health image). *P<0.05, **P<0.01. D–G, Immunofluorescent images of MYO (D), intramyocardial microvessel (E and F), and epicardial normal coronary artery segment (G) from case of DCM. ACE2 (red) and TMPRSS2 (purple) were stained with connexin 43 (Cx43; MYO marker), CD146 (PC marker), and CD31 (EC marker; all in green). Microvessels are outlined with a white-dotted border (E and F). H–I, Relative ACE2 (H) and TMPRSS2 (I) expression among MYO, pericyte (PC), and vascular endothelial cell (EC) from normal and DCM hearts. Four different fields were selected, and the percentage of ACE2 and TMPRSS2 positive areas in each cell type were analyzed by Image J. Relative ACE2 and TMPRSS2 expressions were adjusted to the level in MYO (normal heart). **P<0.01 vs MYO. ††P<0.01 vs PC. ‡‡P<0.01 vs EC (micro). J, Percent of cases showing retrieval of SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR) of the heart and lung samples in 15 COVID-19 cases. K, Viral copies number/mg tissue in 14 lungs and 1 left atrium. L–N, COVID-19 autopsy heart (80-y-old male). SARS-CoV-2 virus was positive in left atrium by RT-PCR. H&E sections did not show any virus-related myocardial damage/inflammation (L). M, Showed corresponding image of virus RNA scope in-situ hybridization. SARS-CoV-2 viral RNA positive cells were visualized by the colocalization of SARS-CoV-2 positive-sense (genomic; green) and negative-sense (replicative intermediate; red) probe (white arrow heads). High-power image of white-rectangle field in (M) showed SARS-CoV-2 replication in cardiomyocytes, which showed Cx43 positive (purple) by additional staining (N). O–Q, COVID-19 autopsy lung (80-y-old female). Thickened alveolar wall with inflammatory cells, including multinuclear giant cells and the hyaline membranes in alveolar space was shown in H&E (O), representing diffuse alveolar damage. Corresponding image of viral RNA scope (P) with multiple double-positive cells (green and red). White-rectangle area in (P) showed infected reactive pneumocyte (Q). R, Number of viral positive cells/mm2 field analyzed by ZEN (Zeiss). Bar graphs (K, R) were expressed in median (interquartile range).In one report, evidence of SARS-CoV-2 detected by real-time reverse transcription polymerase chain reaction of heart in cases of clinically suspected myocarditis or new onset heart failure was quite rare, occurring in 5%.4 In another report of 39 SARS-CoV-2 autopsies, 61.5% showed positive SARS-CoV-2 in the heart5; however, none of them had clinical myocarditis, but the virus could be located in the heart by ISH in interstitial cells or macrophages.5 Combined with our findings, even in the presence of SARS-CoV-2 in the heart, our data as well as that of others suggest viral myocarditis is quite rare and likely not a major cause of myocardial injury.Nonstandard Abbreviations and AcronymsACE2angiotensin-converting enzyme 2COVID-19coronavirus disease 2019ISHin-situ hybridizationSARS-severe acute respiratory syndrome CoV-2 coronavirus 2TMPRSS2transmembrane serine protease 2DisclosuresCVPath Institute has received institutional research support from R01 HL141425 Leducq Foundation Grant; 480 Biomedical; 4C Medical; 4Tech; Abbott; Accumedical; Amgen; Biosensors; Boston Scientific; Cardiac Implants; Celonova; Claret Medical; Concept Medical; Cook; CSI; DuNing, Inc; Edwards LifeSciences; Emboline; Endotronix; Envision Scientific; Lutonix/Bard; Gateway; Lifetech; Limflo; MedAlliance; Medtronic; Mercator; Merill; Microport Medical; Microvention; Mitraalign; Mitra assist; NAMSA; Nanova; Neovasc; NIPRO; Novogate; Occulotech; OrbusNeich Medical; Phenox; Profusa; Protembis; Qool; Recor; Senseonics; Shockwave; Sinomed; Spectranetics; Surmodics; Symic; Vesper; W.L. Gore; Xeltis. A.V. Finn is supported by AWS COVID-19 Diagnostic Development Initiative; has received honoraria from Abbott Vascular; Biosensors; Boston Scientific; Celonova; Cook Medical; CSI; Lutonix Bard; Sinomed; Terumo Corporation; and is a consultant to Amgen; Abbott Vascular; Boston Scientific; Celonova; Cook Medical; Lutonix Bard; Sinomed. A. Cornelissen receives research grants from University Hospital RWTH Aachen. R. Virmani has received honoraria from Abbott Vascular; Biosensors; Boston Scientific; Celonova; Cook Medical; Cordis; CSI; Lutonix Bard; Medtronic; OrbusNeich Medical; CeloNova; SINO Medical Technology; ReCore; Terumo Corporation; W. L. Gore; Spectranetics; and is a consultant for Abbott Vascular; Boston Scientific; Celonova; Cook Medical; Cordis; CSI; Edwards Lifescience; Lutonix Bard; Medtronic; OrbusNeich Medical; ReCore; Sinomededical Technology; Spectranetics; Surmodics; Terumo Corporation; W. L. Gore; Xeltis. The other authors declare no conflicts.Footnotes*These authors contributed equally to this article.This manuscript was sent to William C. Sessa, Senior consulting editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page 543.Correspondence to: Aloke V. Finn, MD, CVPath Institute, Inc, 19 Firstfield Rd, Gaithersburg, MD 20878. Email [email protected]org