Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte

D’Onofrio, Nunzia; Scisciola, Lucia; Sardu, Celestino; Trotta, Maria Consiglia; De Feo, Marisa; Maiello, Ciro; Mascolo, Pasquale; De Micco, Francesco; Turriziani, Fabrizio; Municinò, Emilia; Monetti, Pasquale; Lombardi, Antonio; Napolitano, Maria Gaetana; Marino, Federica Zito; Ronchi, Andrea; Grimaldi, Vincenzo; Hermenean, Anca; Rizzo, Maria Rosaria; Barbieri, Michelangela; Franco, Renato; Campobasso, Carlo Pietro; Napoli, Claudio; Municinò, Maurizio; Paolisso, Giuseppe; Balestrieri, Maria Luisa; Marfella, Raffaele

doi:10.1186/s12933-021-01286-7

Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte

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Published: 07 May 2021

Volume 20, article number 99, (2021)
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Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte

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Nunzia D’Onofrio¹^na1,
Lucia Scisciola²^na1,
Celestino Sardu ORCID: orcid.org/0000-0001-5099-3790²,
Maria Consiglia Trotta³,
Marisa De Feo⁴,
Ciro Maiello⁵,
Pasquale Mascolo⁶,
Francesco De Micco⁶,
Fabrizio Turriziani²,
Emilia Municinò⁷,
Pasquale Monetti⁷,
Antonio Lombardi⁷,
Maria Gaetana Napolitano⁷,
Federica Zito Marino⁸,
Andrea Ronchi⁸,
Vincenzo Grimaldi²,
Anca Hermenean⁹,
Maria Rosaria Rizzo²,
Michelangela Barbieri²,
Renato Franco⁸,
Carlo Pietro Campobasso⁶,
Claudio Napoli²,
Maurizio Municinò⁷,
Giuseppe Paolisso^2,10,
Maria Luisa Balestrieri¹ &
…
Raffaele Marfella^2,10

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Abstract

Rationale

About 50% of hospitalized coronavirus disease 2019 (COVID-19) patients with diabetes mellitus (DM) developed myocardial damage. The mechanisms of direct SARS-CoV-2 cardiomyocyte infection include viral invasion via ACE2-Spike glycoprotein-binding. In DM patients, the impact of glycation of ACE2 on cardiomyocyte invasion by SARS-CoV-2 can be of high importance.

Objective

To evaluate the presence of SARS-CoV-2 in cardiomyocytes from heart autopsy of DM cases compared to Non-DM; to investigate the role of DM in SARS-COV-2 entry in cardiomyocytes.

Methods and results

We evaluated consecutive autopsy cases, deceased for COVID-19, from Italy between Apr 30, 2020 and Jan 18, 2021. We evaluated SARS-CoV-2 in cardiomyocytes, expression of ACE2 (total and glycosylated form), and transmembrane protease serine protease-2 (TMPRSS2) protein. In order to study the role of diabetes on cardiomyocyte alterations, independently of COVID-19, we investigated ACE2, glycosylated ACE2, and TMPRSS2 proteins in cardiomyocytes from DM and Non-DM explanted-hearts. Finally, to investigate the effects of DM on ACE2 protein modification, an in vitro glycation study of recombinant human ACE2 (hACE2) was performed to evaluate the effects on binding to SARS-CoV-2 Spike protein. The authors included cardiac tissue from 97 autopsies. DM was diagnosed in 37 patients (38%). Fourth-seven out of 97 autopsies (48%) had SARS-CoV-2 RNA in cardiomyocytes. Thirty out of 37 DM autopsy cases (81%) and 17 out of 60 Non-DM autopsy cases (28%) had SARS-CoV-2 RNA in cardiomyocytes. Total ACE2, glycosylated ACE2, and TMPRSS2 protein expressions were higher in cardiomyocytes from autopsied and explanted hearts of DM than Non-DM. In vitro exposure of monomeric hACE2 to 120 mM glucose for 12 days led to non-enzymatic glycation of four lysine residues in the neck domain affecting the protein oligomerization.

Conclusions

The upregulation of ACE2 expression (total and glycosylated forms) in DM cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favouring the cellular entry of SARS-CoV2.

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The potential translational impact of the study results

In patients with diabetes mellitus, the upregulation of ACE2 expression in cardiomyocytes, together with non-enzymatic glycation favouring protein oligomerization, could increase the susceptibility to COVID-19 infection and worse prognosis. However, the control of the expression of ACE2 and its glycated form could represent a therapeutic target to prevent COVID-19 infection and worse prognosis in patients with diabetes.

Introduction

The coronavirus disease-19 (COVID-19), caused by the RNA single-stranded enveloped virus of severe acute respiratory syndrome (SARS)-CoV-2, has a significant impact on the cardiovascular (CV) system by direct myocardial damage [1]. Indeed, a considerable number of hospitalized COVID-19 patients could develop cardiac injury (24.4%), with a consequent higher rate of mortality (72.6%), [2]. Notably, among hospitalized COVID-19 patients with diabetes (DM), about half of them developed myocardial damage [3]. Indeed, DM is very common among hospitalized COVID-19 patients, has a significant impact on the treatment [4], and negatively influences clinical outcomes in affected patients [5,6,7]. However, specific therapies to prevent coagulopathies, over-inflammation, and hyperglycemia may represent a valid therapeutic option for treating asymptomatic and non-critically ill COVID-19 patients with DM as critically-ill DM patients [8,9,10,11,12].

In this context, the impact of hyperglycemia in the progression and deterioration of heart function in COVID-19 patients is currently of great importance. Indeed, in DM patients, the severity of SARS-CoV-2 infection has been attributed to impaired innate and adaptive immunity, upregulation of ACE2, and potential changes in the glycation of ACE2 [4, www.uniprot.org; entry: Q9BYF1, entry name: ACE2_HUMAN) showing in red the glycated lysine residues obtained after 12 days of incubation with 120 mM of glucose. b Position glycated lysine (K) after 12 days of incubation with 12 mM, 60 mM, and 120 mM of glucose and function of glycated sites. c Human ACE2 homodimer (PDB 1r42) showing the lysine 353 (K353), involved in the Spike-RBD binding to ACE2, lysine 470 (K470) (unknown function). ACE2 structure from PDB 6M17 showing the glycated lysine 619 (K619), 631 (K631), 659 (K659), and 689 (K689) in the polar neck region involved in the dimerization of ACE2

Full size image

Results showed that among the seven glycated residues, only lysine 353 (Lys 353) (Fig. 4c) has previously been reported as important for binding Spike-RBD [31]. However, under our experimental conditions, minor glycation of Lys 353 (0.6%) was found. Also, in the hACE2 (Protein Data Bank, PDB 1r42), the Lys 470 with unknown function displayed a 4.98% of glycation at 120 mM of glucose (Fig. 4c). Notably, a higher number of glycated residues were detected in the polar region of ACE2 involved in the dimerization (neck domain), with Lys 619, Lys 631, Lys 659, and Lys 689 showing 1.47%, 5.28%, 7.62%, and 6.78% of glycation, respectively (Fig. 4c). The structures shown in Fig. 4c are from PDB 6M17. Next, we verified that the glycosylation-associated structural heterogeneity of hACE2 was quite difficult to eliminate. ACE2 glycosylation was removed by overnight incubation with PNGase F. SDS-PAGE results showed that the deglycosylated hACE2 has a lower apparent molecular weight due to the PNGase digestion. However, its band is still heterogeneous with a diffused aspect (Additional file 1: Figure SIX).

For SPR measurements of glycated ACE-SARS-CoV-2 Spike protein binding, an affinity coupling approach using a biosensor functionalized with an anti-human F.C. antibody immobilization kit (Cytiva, catalog no. BR-1008-39) was used. This system allowed to bind the Spike protein via its F.C. affinity tail with chip recycling for the whole study. The system showed to be functional, and an amount of 1 µg/assay of immobilized Spike protein was found to work optimally. In line with the low grade of glycation found at the Lys 353 located in the hACE2 domain of binding for SARS-CoV-2 Spike, results showed a minimal influence of glycation on the binding properties to SARS-CoV-2 Spike, as indicated by the K_a and K_d and the protein ligand-analyte affinity (K.D.) (9.69 nM in control and 11.07 nM in glycated hACE2 with 120 mM of glucose) (Additional file 1: Figure SX).

Glycation modifies ACE migration

The impact of the glycated residues detected in the neck domain of ACE2 on the protein structure was further investigated by SDS-PAGE under reducing and non-reducing conditions. To this end, the oligomerization state of hACE2 and SARS-CoV-2 Spike protein was first evaluated in samples before starting mild glycation experiments and binding measurement, confirming the purity of dimeric spike and the monomeric hACE2 protein (detected band at 100 kDa) used in this study (Fig. 5a). Notably, the SDS-PAGE of glycated hACE2 (Glyc-hACE) (120 mM glucose) showed important differences compared to non-glycated hACE2 (hACE) (Fig. 5b). In a non-reducing setting, hACE showed only one band at 100 kDa, whereas the Glyc-hACE showed both a predominant band over 100 kDa along with another band at the higher molecular weight (250 kDa). Under reducing conditions, both hACE and Glyc-hACE showed the predominant band of at 100 kDa. However, despite the experimental reducing conditions, a weak band at 250 kDa was still observed for Glyc-hACE. The evidence of glycation role on ACE2 dimer formation under a non-reducing setting (p < 0.05) was provided by the quantitative ratio of the dimeric to monomeric form (Fig. 5c).

These results indicated the mild glycation modified hACE2 protein migration and oligomerization process, supporting non-enzymatic glycation at neck domain level on hACE2 oligomerization and dimer formation (Addition file 2).

Discussion

For the first time in literature, our investigation provides a new clinical human and an “in vitro-ex vivo” post-mortem model to evaluate the SARS-COV-2 ligand with ACE2 receptor, its entrance, and replication in cardiomyocytes in patients with normoglycemia, and under a condition of over-glycaemic stress. Notably, in the COVID-19 autopsy cohort, the DM vs. Non-DM specimens had a significantly higher number of SARS-COV-2 localized inside the cardiomyocytes. Thus, we evidenced, by immunofluorescence analysis of cardiac specimens, a higher expression of myocardial ACE2 protein in DM vs. Non-DM. Notably, DM sections mainly evidenced a peri-nuclear ACE2 localization. We also observed in COVID-19 patients an increase of ACE2 in heart specimens from DM as compared to Non-DM patients. In this setting, in the immunoblotting analysis of explanted hearts from non-COVID-19 patients, we found an increase of glycosylated ACE2 and TMPRSS2 protein content in DM vs. Non-DM specimens. Notably, it was highest in COVID-19 myocardial tissue of DM vs. Non-DM. Furthermore, the ratio between glycosylated on total ACE2, confirmed the more consistent expression levels of glycosylated ACE2 protein in COVID-19 patients, with the highest upregulation in DM vs. Non-DM (Additional file 3).

From current literature, authors reported that SARS-CoV-2 could infect human cardiomyocytes in culture and different models of cardiac tissue [31,32,33]. On the other hand, these authors did not provide any evidence about cardiomyocyte SARS-CoV-2 infection in the subgroup of high-risk patients, such as DM patients. Secondly, they did not assess the specific pathways induced by DM to promote the virus entry into the cardiomyocyte. In this regard, the heart shows a higher expression of the ACE2 receptor, which could mediate SARS-CoV-2 cell entry [34]. Notably, during SARS-COV-2 infection, the trimeric Spike protein is cleaved into S1 and S2 subunits with the S1 containing the RBD that directly binds to the peptidase domain ACE2 [34]. The binding kinetics between the SARS-CoV-2 spike protein and the ACE2 receptor depends on protein structures, and their molecular interactions are altered by glycosylation [35,36,37]. In particular, in the SARS-CoV-2/RBD–ACE2 complex structure, the glycan–RBD interaction has important roles in the binding [37]. Indeed, it is a chain of Asn90-linked NAG–NAG–β-d-mannose in contact with the Thr402 of the SARS-CoV-2/RBD [38]. In this context, we observed a consistent increase of ACE2 protein expression in heart tissue of DM vs. Non-DM patients (p < 0.0032). However, to elucidate the supposed model of intra-cellular entrance and replication of SARS-COV-2, in the in vitro model, we evaluated the ACE2 glycation and binding to SARS-CoV-2 Spike. Thus, we detected the ACE2 glycation after 12 days of incubation with glucose for all three concentrations tested (12, 60, and 120 mM). Notably, despite the minimal influence of glycation on the binding properties to SARS-CoV-2 Spike, we evidenced the effect of chemical modification on the ACE2 oligomerization state. However, the mild glycation modified ACE2 protein migration and oligomerization process could support non-enzymatic glycation at neck domain level on hACE2 oligomerization and dimer formation. Furthermore, there is a higher expression of glycosylated ACE2 in heart samples from DM vs. Non-DM patients (p < 0.05), with more consistent levels in COVID-19 patients with DM (p < 0.01). To date, this result could support the existing relationship between DM and the upregulation of glycosylated ACE2 protein. Again, COVID-19 patients with DM also showed higher levels of TMPRSS2 expression. This study result could confirm the hypothesis that the glycosylation of ACE2 could be linked to a higher expression of TMPRSS2 protein [39].

Therefore, in DM vs. Non-DM patients, the upregulation of ACE2 expression (total and glycosylated forms) in cardiomyocytes, along with non-enzymatic glycation, could increase the susceptibility to COVID-19 infection in DM patients by favoring the cellular entry of SARS-CoV2. In this context, the endothelial and platelet cells pathology, and fibrin(ogen) over-synthesis could negatively interact with SARS-CoV-2 in DM vs. Non-DM patients. Indeed, in DM patients there is a higher rate of clotting in COVID-19 patients [8], that is linked to a prominent elevation of fibrinogen and d-dimer/fibrin(ogen) degradation products, causing a hypercoagulability [40]. Notably, the degree of d-dimer elevation positively correlated with mortality in COVID-19 patients [40]. In addition to arterial thrombotic events and microvascular thrombotic disorders, COVID-19 patients often have mild thrombocytopenia and appear to have increased platelet consumption, together with a corresponding increase in platelet production [41]. Conversely, if the entry of the SARS-COV-2 into the host cell requires the cleavage of the S protein into the S1 and S2 subunits by TMPRSS2 to promote pathogenicity and myocardial damage reported in COVID-19 patients [19, 20], we might speculate that DM vs. Non-DM might over-express these adverse clinical pathways. Therefore, all these pathological processes might be over-expressed in DM vs. Non-DM, and mainly induced by SARS-CoV-2 in DM vs. Non-DM patients. To date, this is in line with our study results. Indeed, we evidenced, (i) a significantly higher number of cardiomyocytes with SARS-COV-2 localization (34.7 ± 5.3% vs. 14.3 ± 4.1%, P = 0.001) in the DM vs. Non-DM specimens (Fig. 1); (ii) a consistent increase of myocardial ACE2 protein expression in DM patients compared to Non-DM subjects in both explanted hearts from COVID-19 patients and controls; (iii) a higher glycosylated ACE2 and TMPRSS2 protein content in the heart from DM compared with Non-DM patients in both explanted hearts from COVID-19 patients and controls (Fig. 3b, c); (iv) that COVID-19 myocardial tissue from DM patients had a more pronounced TMPRSS2 protein expression level compared to Non-DM (p < 0.01) (Fig. 3d, e). However, we might suggest that all these pathogenic mechanisms over-expressed in DM vs. Non-DM could promote pathogenicity and myocardial damage in controls and much more in a condition of SARS-CoV-2 infection.

Thus, here we hypothesized that increased glycosylated ACE2 and TMPSS in cardiomyocytes, due to DM milieu, might impact the SARS-COV-2 cells entry. However, this might represent a crucial pathophysiological step, leading to worse clinical outcomes in COVID-19 patients with DM.

Although it is well recognized that ACE2 glycation is linked to a DM condition, the pathways that promote the entry of SARS-COV-2 in the cells are determined by spike glycation [41]. The mechanism by which DM milieu may be involved in this process is not fully clarified. Thus, to elucidate this aspect, we conducted an in vitro study to evaluate the occurrence of non-enzymatic glycation of ACE2 and its possible pathogenic role in the changing of the protein binding to SARS-CoV-2 Spike or the protein structure in terms of the amino acid (mostly lysine) glycation. However, the evaluation of the functional impact of non-enzymatic glycation on ACE2 [37,38,39,40] showed seven glycated residues of which only one (Lys 353) in the important for the binding domain for Spike-RBD, and four (Lys 619, Lys 631, Lys 659, and Lys 689) in the neck domain involved in ACE2 dimerization [36,37,38,39,40,41,42]. In line with the low percentage of glycation detected at Lys 353, we found that the mild glycation of ACE2 monomer slightly influenced the binding properties to Spike protein. Then, the evaluation of the oligomerization state of ACE2 after in vitro mild non-enzymatic glycation, besides the predominant band of monomeric ACE2 (100 kDa), unveiled the occurrence of a band at 250 kDa under non-reducing conditions. This supports the occurrence of changes in the oligomerization process towards dimer formation. Indeed, under reducing conditions, the glycated ACE2 showed an increase in the band at 100 kDa and decreased in the band at 250 kDa. Although the band at 250 is detectable in our experimental conditions in small amounts, it cannot exclude that the long-term effect of diabetes on glycation could significantly influence the oligomerization favoring dimeric ACE2. This evidence could reflect the highest susceptibility of DM to SARS-CoV-2 infections because oligomerization increases the binding affinity and avidity to SARS-CoV-2 Spike protein. Indeed, as recently demonstrated, the trimeric engineered ACE2 variant has a binding affinity of ~ 60 pM for SARS-CoV-2 Spike protein, compared with 77–90 nM for monomeric ACE2 and 12–22 nM for dimeric ACE2 constructs [16, 18, 43]. Finally, although dimeric ACE2 is unlikely to engage more than one RBD from the same spike protein, it cannot be ruled out that higher levels of ACE2 dimers on the cell surface might further cluster to induce more RBDs. Then, the RBDs could adopt up conformation and help SARS-CoV-2 to transit from the pre-fusion state to the post-fusion state. However, in addition to the known adverse effects of hyperglycemia in patients with COVID-19 [43,44,45], here we reported the enhanced expression of glycosylated ACE2 in DM cardiomyocytes.

Furthermore, this result provides the first evidence on the in vitro glycation of ACE2 at the neck domain of dimerization. Indeed, the enhanced ACE2 glycation suggests that this system's activation by DM milieu is associated with an increased ACE2 oligomerization and avidity for SARS-COV-2 Spike binding. Finally, these effects could favor cardiomyocyte rather than interstitial or endothelial cell virus invasion (Fig. 6).

On the other hand, because of data from autopsied cases, we cannot exclude that post-mortem condition may influence the expression of ACE2 and TMPSS, such as observed in DM patients. Thus, to exclude possible post-mortem condition-induced changes, we evaluated the effects of DM milieu on ACE2 and TMPSS expression in the explanted hearts. Moreover, according to the autopsied data, we evidenced an increase of glycosylated ACE2 and TMPRSS2 protein content in DM specimens compared to Non-DM specimens from non-COVID-19 hearts (Additional file 4).

Conclusion

Furthermore, taken together all these data might suggest that DM might promote the ACE2 modifications, favoring SARS-COV-2 entry in cardiomyocytes, independently of both post-mortem and COVID-19 molecular changes. Thus, we might speculate that increased ACE2 glycation and TMPSS expression in cardiomyocytes, as a consequence of DM, may favor SARS-COV-2 entry in the host cell. Therefore, this could represent a crucial pathophysiological step leading to worse clinical outcomes and cardiovascular events in COVID-19 patients with DM [46].

Availability of data and materials

Data and materials are available if required.

Abbreviations

ADA:: American Diabetes Association
COVID-19:: Coronavirus disease-19
CV:: Cardiovascular
DM:: Diabetes mellitus
FFPE:: Formalin-fixed paraffin-embedded
hACE2:: Recombinant human ACE2
HTX:: Heart transplantation
ISH:: In situ hybridization
Non-DM:: Without diabetes mellitus
PBS:: Phosphate buffered saline
RBD:: Spike receptor‐binding domain
RT:: Room temperature
SARS-CoV-2:: Severe acute respiratory syndrome
SPR:: Surface plasmon resonance
TBS:: Tris-buffered saline
TMPRSS2:: Transmembrane protease serine protease-2

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Acknowledgements

R.M is the guarantor of the presented data. Authors fully and equally contributed to this research.

Funding

This work was supported by VALERE 2019 Program, University of Campania L. Vanvitelli.

Author information

Nunzia D’Onofrio and Lucia Scisciola equally contributed as the author

Authors and Affiliations

Department of Precision Medicine, University of Campania L. Vanvitelli, Naples, Italy
Nunzia D’Onofrio & Maria Luisa Balestrieri
Department of Advanced Medical and Surgical Sciences, University of Campania L. Vanvitelli, Piazza Miraglia, 2, 80138, Naples, Italy
Lucia Scisciola, Celestino Sardu, Fabrizio Turriziani, Vincenzo Grimaldi, Maria Rosaria Rizzo, Michelangela Barbieri, Claudio Napoli, Giuseppe Paolisso & Raffaele Marfella
Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”, Naples, Italy
Maria Consiglia Trotta
Department of Cardio-Thoracic Sciences, University of Campania “Luigi Vanvitelli”, Naples, Italy
Marisa De Feo
Unit of Cardiac Surgery and Transplants, AORN Ospedali dei Colli-Monaldi Hospital, 80131, Naples, Italy
Ciro Maiello
Department of Experimental Medicine Forensic Pathology Service, University of Campania L. Vanvitelli, Naples, Italy
Pasquale Mascolo, Francesco De Micco & Carlo Pietro Campobasso
Department of Forensic, Evaluative and Necroscopic Medicine, ASL Napoli 2 NORD, Naples, Italy
Emilia Municinò, Pasquale Monetti, Antonio Lombardi, Maria Gaetana Napolitano & Maurizio Municinò
Department of Mental and Physical Health and Preventive Medicine, University of Campania L. Vanvitelli, Naples, Italy
Federica Zito Marino, Andrea Ronchi & Renato Franco
Institute of Life Science, Vasile Goldis Western University, Arad, Romania
Anca Hermenean
Mediterranea Cardiocentro, Naples, Italy
Giuseppe Paolisso & Raffaele Marfella

Authors

Nunzia D’Onofrio
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Lucia Scisciola
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Celestino Sardu
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Maria Consiglia Trotta
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Marisa De Feo
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Ciro Maiello
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Francesco De Micco
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Fabrizio Turriziani
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Emilia Municinò
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Pasquale Monetti
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Antonio Lombardi
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Federica Zito Marino
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Andrea Ronchi
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Vincenzo Grimaldi
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Anca Hermenean
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Maria Rosaria Rizzo
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Michelangela Barbieri
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Renato Franco
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Carlo Pietro Campobasso
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Claudio Napoli
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Maurizio Municinò
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Giuseppe Paolisso
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Maria Luisa Balestrieri
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Contributions

NDO, LS: study design; CS, RM, MLB, GP: study editing; MCT, EM, PM, AL, MGN, FZM, AR, VG: study experiments; RM, CN, GP and MLB: study conception and design; FT, PM, FDM, AH: data collection; MDF, CM: heart transplant; MRR, MB: study revision; RF, CPC, MM: histopathological analysis of heart specimens. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Celestino Sardu.

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The local ethics committee approved this study of the Vanvitelli University. The participants signed informed written consent to participate in this study.

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There are not competing interests to declare.

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Supplementary Information

Additional file 1

: Figure SI. Immunofluorescence negative control. Sections from diabetic and non-diabetic patients were performed with blocking solution, supplemented with a non-immune immunoglobulin IgG antibody, following by secondary antibody Alexa Fluor 488 or 633 incubation for 1 hour at RT. All samples were stained with DAPI (5 μg/ml) for 10 min before mounting in Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA). All slides were imaged using a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany) with a plan apochromat X63 (NA1.4) oil immersion objective. DM: diabetes mellitus; Non-DM: without diabetes mellitus. Table I: Mass accuracy and retention time (RT) of hACE2 tryptic peptides targeted by glycation. The measurement accuracies were reported in parts per million (ppm) and the chromatographic retention times in minutes (mins). Figure SII: Mass spectra of the triply charged hACE2 tryptic peptide 342-357 in the glycated (panel A) and non-glycated (panel B) form. Figure SIII: Mass spectra of the triply charged hACE2 tryptic peptide 466-475 in the glycated (panel A) and non-glycated (panel B) form. Figure SIV: Mass spectra of the triply charged hACE2 tryptic peptide 560-577 in the glycated (panel A) and non-glycated (panel B) form. Figure SV: Mass spectra of the triply charged hACE2 tryptic peptide 601-621 in the glycated (panel A) and non-glycated (panel B) form. Figure SVI: Mass spectra of the triply charged hACE2 tryptic peptide 626-644 in the glycated (panel A) and non-glycated (panel B) form. Figure SVII: Mass spectra of the triply charged hACE2 tryptic peptide 658-671 in the glycated (panel A) and non-glycated (panel B) form. Figure SVIII: Mass spectra of the triply charged hACE2 tryptic peptide 679-697 in the glycated (panel A) and non-glycated (panel B) form. Figure SIX: hACE2 PNGase F deglycosylation. SDS-PAGE performed on 500 nanograms of hACE2 digested with five units of PNGase F (Promega, catalog n. V4831) overnight at 37 °C and then analyzed on a NuPAGE 4-12 Bis-Tris gel (Thermo, catalog no. NP326) using a NuPAGE MES running buffer (Thermo, catalog no. NP0002). Lane 1=Bio-Rad Precision Standard (catalog no. 161-0373); lane 2=deglycosylation blank; lane 3=PNGase-digested hACE2; lane 4= undigested hACE2. The band at about 35 KDa in lanes 2 and 3 is the PNGase F. Figure SX. Glycated hACE binding to SARS-CoV-2 Spike protein. A, hACE2 control, B, hACE2 at 12 days incubation time with 60 mM and C, 120 mM of glucose. D, Values of kinetic binding parameters of hACE2-SARS-CoV-2 Spike protein obtained from SPR measurement. Binding sensorgrams for SARS-CoV-2 Spike protein and several analyte concentrations of 1 µg of immobilized Spike protein and ACE2. Samples of ACE2 were diluted in HBS-EP+ at different concentration (64 µg/ml, 32µg/ml, 16 µg/ml, 8 µg/ml, and 4 µg/ml) and injected for 120s at a flow rate of 30µl/min on flow cell 3 and 4. Formulation buffer was run as a control. Dissociation was followed for 300sec; regeneration was achieved with a 60-sec pulse of 3M MgCl2. ACE2 concentrations were listed on the right side and were expressed in µg/ml. Ligand-analyte affinity (KD) and kinetic parameters (Association rate, Ka; Dissociation rate, Kd) of Originator and Biosimilar Rituximab was calculated with the Biacore T200 Evaluation Software (version 2.0; GE Healthcare). Figure SXI: RBD amino acid sequence; 319–541 residues and crystal structure of SARS-CoV-2 Spike receptor-binding domain (RBD) bound with ACE2 (PDB ID: 6M0J). Lysine 353 (K353) undergoes mild glycation by high-glucose (12 mM) exposure. Asparagine (N).

Additional file 2: Figure S2

. Mass spectra of the triply charged hACE2 tryptic peptide 342-357 in the glycated (panel A) and non-glycated (panel B) form. hACE2 is for human Angiotensin Converting enzyme 2 type.

Additional file 3: Figure S3

. Mass spectra of the triply charged hACE2 tryptic peptide 466-475 in the glycated (panel A) and non-glycated (panel B) form. hACE2 is for human Angiotensin Converting enzyme 2 type.

Additional file 4: Figure S4

. Mass spectra of the triply charged hACE2 tryptic peptide 560-577 in the glycated (panel A) and non-glycated (panel B) form. hACE2 is for human Angiotensin Converting enzyme 2 type.

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D’Onofrio, N., Scisciola, L., Sardu, C. et al. Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte. Cardiovasc Diabetol 20, 99 (2021). https://doi.org/10.1186/s12933-021-01286-7

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Received: 19 March 2021
Accepted: 23 April 2021
Published: 07 May 2021
DOI: https://doi.org/10.1186/s12933-021-01286-7

Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte