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

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.

Keywords: ACE2; COVID-19; Cardiomyocyte; Diabetes; Heart; SARS-CoV-2.

Fig. 1

SAR-COV-2 in myocardial tissue from COVID-19 autopsies. a Representative myocardial tissue specimen from 60 patients without diabetes (Non-DM) (× 400). b Representative myocardial tissue specimens from 37 patients with diabetes (DM). Brown punctate evidenced the SARS-COV-2 RNA copies in the cardiomyocytes (96 positive cells/237 cells) (× 400). These structures are marked with black arrows (SARS-COV2), and with blue arrows (Cardiomyocytes). c Mean ± SD of the percentage of SARS-COV-2 positive cardiomyocyte. Statistical test: Student’s t-test. Bonferroni correction was used to make pairwise comparisons. *P < 0.05

Fig. 2

ACE2 immunofluorescence detection. a Representative images of ACE2 expression (red) and cardiac troponin T (green) in non-COVID-19 and COVID-19 heart tissue from patients with diabetes (DM) and patients without diabetes (Non-DM). Cell nuclei were stained blue with DAPI. b Fluorescence intensity analysis in the Non-DM Non-COVID-19 (n = 43) versus DM Non-COVID-19 (n = 7), (p = 0.0032) and Non-DM COVID-19 (n = 17) versus DM COVID-19 (n = 30), (p = 0.009) of myocardial ACE2 expression was estimated with Image J software. Analysis comparing DM COVID-19 versus Non-DM Non-COVID-19 (p = 8.96865E−05) and DM COVID-19 versus DM Non-COVID-19 (p = 1.10E−04) was also reported. Shown as mean ± SD. Statistical test: Student’s t-test. Bonferroni correction was used to make pairwise comparisons. Data were presented as box and whisker plots showing medians (middle line) and in boxes the third and first quartiles (75th and 25th percentiles), while the whiskers show 1.5 × the interquartile range (IQR) above and below the box. Scale bar = 10 µm. ACE2 angiotensin-converting enzyme 2

Fig. 3

Glycosylated ACE2 and TMPRSS2 protein levels. a–c, Representative images and bar graph of Western blotting analysis (n = 4) of glycosylated (Glyc) ACE2, total ACE2 and Glyc/total ACE2 ratio in heart tissue from patients without diabetes (Non-DM) Non-COVID-19 (n = 43) versus heart tissue from patients with diabetes (DM) Non-COVID-19 (n = 7), (p = 0.03276 and p = 0.047, respectively) and Non-DM COVID-19 (n = 17) versus DM COVID-19 (n = 30), (p = 0.002391 and p = 0.0025, respectively). Shown as mean ± SD. Statistical test: Student’s t-test. Bonferroni correction was used to make pairwise comparisons. d, e Representative images and bar graph of Western blotting analysis (n = 3) of TMPRSS2 in Non-DM Non-COVID-19 (n = 43) versus DM Non-COVID-19 (n = 7), (p = 0.0344) and Non-DM COVID-19 (n = 17) versus DM COVID-19 (n = 30), (p = 0.001) heart samples. Protein expression was determined by ImageJ 1.52n software and quantified using α-tubulin or GAPDH. Values are expressed as arbitrary units (A.U.). Shown as mean ± SD. Statistical test: See (a–c). *p < 0.05 vs. non-DM (Non-COVID-19); ^§p < 0.01 vs. non-DM (COVID-19). ACE2 angiotensin-converting enzyme 2, TMPRSS2 transmembrane protease serine 2, GAPDH glyceraldehyde 3-phosphate dehydrogenase

Fig. 4

Mapping glycation on hACE2. a Human ACE2 sequence (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

Fig. 5

Effect of glycation on hACE2 migration. a SDS-PAGE was conducted using hACE2 and SARS-CoCOVV-2 Spike protein aliquots collected before starting glycation and SPR measurements. b SDS-PAGE was conducted using 8% gels in reducing and non-reducing conditions with hACE2 incubated for 12 days with glucose 120 mM (Glyc hACE2). Molecular weight indicators are displayed at the center. c Ratio of the dimeric to monomeric form of ACE2 in reducing and non-reducing condition. *p < 0.05 vs. non glycated ACE2. Shown as mean ± SD. Statistical test: Student’s t-test

Fig. 6

Schematic representation of the proposed effect of diabetes milieu on ACE2 in the heart of patients with type 2 diabetes. In patients with diabetes, the enhanced long-term non-enzymatic glycation of ACE2 at the neck domain of dimerization can affect ACE2 oligomerization and, consequently, its avidity for SARS-COV-2 Spike binding, potentially favoring cardiomyocyte virus entry