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Additional file 1: Figure S1. Western blot analysis of GlycACE2. Representative immunoblotting of recombinant hACE2 protein (ab151852, Abcam) after in vitro long-term exposure to glucose 120 mM was separated on SDS-PAGE by using 7% gels in reducing and non-reducing conditions and then transferred on nitrocellulose membrane. Membrane was incubated with specific primary antibody against GlycACE2 (1:1000) (#4355, Cell Signaling Technology). Molecular weight indicators are displayed at the center. Figure S2. Ejection fraction, TAPSE, and the E/e′ at week 1 (Basal) and week 48 (follow-up) in nondiabetic and diabetic patients treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). Data are mean ± SD. *P < 0.05 vs non-diabetics, §P < 0.05 vs basal values. Figure S3. GlycACE2 at week 1 (Basal) and week 48 (Follow-up) in the diabetic patients with good glycemic control (HbA1c < 7%) and diabetic patients with poor glycemic control (HbA1c ≥ 7%) treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). Data are mean ± SD. *P < 0.05 vs non-diabetics, §P < 0.05 vs basal values. Figure S4. A Angiotensin-1–9 (Ang-1–9), Ang 1–7, Mas receptor (MasR), Nuclear factor of activated T-cells (NFAT), in explanted hearts (HTX) at week 1 (Basal) and week 48 (Follow-up) from HTX, in nondiabetic and diabetic patients, treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). B Ang-1–9, Ang 1–7, MasR, and NFAT, in explanted hearts (HTX) at week 1 (Basal) and week 48 (Follow-up) from HTX,in the diabetic patients with good glycemic control (HbA1c < 7%) and diabetic patients with poor glycemic control (HbA1c ≥ 7%) treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). Data are mean ± SD. *P < 0.05 vs non-diabetics, §P < 0.05 vs basal values. Figure S5. Fibrosis percentage in explanted hearts (HTX), at week 1 (Basal) and week 48 (follow-up) from HTX, in the diabetic patients with good glycemic control (HbA1c < 7%) and diabetic patients with poor glycemic control (HbA1c ≥ 7%) treated with ACE-inhibitors (ACE-I) and angiotensin receptor blocker (ARB). Data are mean ± SD. *P < 0.05 vs non-diabetics, §P < 0.05 vs basal values. Table S1. Multivariate linear regression analysis with GlycACE2 as dependent variable.

Background High glycated-hemoglobin (HbA1c) levels correlated with an elevated risk of adverse cardiovascular outcomes despite renin-angiotensin system (RAS) inhibition in type-2 diabetic (T2DM) patients with reduced ejection fraction. Using the routine biopsies of non-T2DM heart transplanted (HTX) in T2DM recipients, we evaluated whether the diabetic milieu modulates glycosylated ACE2 (GlycACE2) levels in cardiomyocytes, known to be affected by non-enzymatic glycosylation, and the relationship with glycemic control. Objectives We investigated the possible effects of GlycACE2 on the anti-remodeling pathways of the RAS inhibitors by evaluating the levels of Angiotensin (Ang) 1–9, Ang 1–7, and Mas receptor (MasR), Nuclear-factor of activated T-cells (NFAT), and fibrosis in human hearts. Methods We evaluated 197 first HTX recipients (107 non-T2DM, 90 T2DM). All patients were treated with angiotensin-converting enzyme inhibitor (ACE-I) or angiotensin receptor blocker (ARB) at hospital discharge. Patients underwent clinical evaluation (metabolic status, echocardiography, coronary CT-angiography, and endomyocardial biopsies). Biopsies were used to evaluate ACE2, GlycACE2, Ang 1–9, Ang 1–7, MasR, NAFT, and fibrosis. Results GlycACE2 was higher in T2DM compared tonon-T2DM cardiomyocytes. Moreover, reduced expressions of Ang 1–9, Ang 1–7, and MasR were observed, suggesting impaired effects of RAS-inhibition in diabetic hearts. Accordingly, biopsies from T2DM recipients showed higher fibrosis than those from non-T2DM recipients. Notably, the expression of GlycACE2 in heart biopsies was strongly dependent on glycemic control, as reflected by the correlation between mean plasma HbA1c, evaluated quarterly during the 12-month follow-up, and GlycACE2 expression. Conclusion Poor glycemic control, favoring GlycACE2, may attenuate the cardioprotective effects of RAS-inhibition. However, the achievement of tight glycemic control normalizes the anti-remodeling effects of RAS-inhibition. Trial registration: https://clinicaltrials.gov/ NCT03546062.