Glycated ACE2 reduces anti-remodeling effects of renin-angiotensin system inhibition in human diabetic hearts

Abstract

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://ichgcp.net/clinical-trials-registry/NCT03546062" title="See in ClinicalTrials.gov">NCT03546062.

Keywords: Diabetes; Diabetic cardiomyopathy; HbA1c; Heart transplantation; RAS-inhibition therapy.

Conflict of interest statement

The authors declare no competing interests.

© 2022. The Author(s).

Figures

Fig. 1

Study protocol

Fig. 2

Flow-chart of the study protocol

Fig. 3

Ejection fraction, tricuspid annular plane systolic excursion (TAPSE) and the ratio of transmitral Doppler early filling velocity to tissue Doppler early diastolic mitral annular velocity (E/e′) and cardiac at week 1 (Basal) and week 48 (follow-up) from HTX with respective Delta values, in nondiabetics and diabetic patient (A), and in diabetic patients in good glycemic control (< 7%) and poor glycemic control (≥ 7%). (Boxplot, a plot type that displays the median, 25th, and 75th percentiles and range). *P < 0.05 vs nondiabetics, §P < 0.05 vs basal values (A). *P < 0.05 vs diabetic patients in good glycemic control (< 7%), §P < 0.05 vs basal values

Fig. 4

ACE2 protein levels in human cardiomyocytes. A Representative images and B analysis of ACE2 expression (red) and Cardiac Troponin T (green) in explanted heart tissue, basal and follow-up biopsies from nondiabetic and diabetic patients with poor or good glycemic control. Cell nuclei were stained blue with DAPI. Microscopy analyses were performed using an LSM 700 confocal microscope (Zeiss, Oberkochen, Germany) with a plan apochromat X63 (NA1.4) oil immersion objective. Fluorescence intensity analysis of myocardial ACE2 expression was estimated with ImageJ software and expressed as arbitrary fluorescence units (AFU). Data are presented as box and whisker plots showing medians (middle line) and the 2.5 and 97.5 percentiles using GraphPad Prism 9.1.2. software. C qRT-PCR analysis of ACE2 mRNA levels, expressed as 2−ΔΔCt ± S.D., in human heart biopsies from nondiabetic patients (blue) and diabetic patients with poor (green) or good (red) glycemic control. *P < 0.05 vs Nondiabetic; ‡P < 0.05 vs Basal biopsy; § vs Diabetic with poor glycemic control at Follow-up biopsy

Fig. 5

ACE2 and GlycACE2 expression in human cardiomyocytes. A Representative images of Western blotting analysis of ACE2 and GlycACE2 in explanted heart tissue, basal and follow-up biopsies from nondiabetic and diabetic patients with poor (HbA1c > 7%) or good (HbA1c < 7%) glycemic control. ACE2 and Glyc ACE2 levels were normalized using α-Tubulin. Glyc ACE2 levels in B nondiabetic and diabetic patients and C diabetic patients with HbA1c < 7% and diabetic patients with HbA1c > 7%. Analysis was performed by ImageJ 1.52n software; values were expressed as percentage and data were shown as box and whisker plots using GraphPad Prism 9.1.2. software. *P < 0.05 vs nondiabetic patients; †P < 0.05 vs diabetic patients in good glycemic control (< 7%), §P < 0.05 vs basal values

Fig. 6

A Evaluation of Angiotensin-1–9 (Ang-1–9), Ang 1–7, MAS1, and NFAT activation molecule 1 (NFAM1) content in explanted hearts (HTX) at week 1 (Basal) and week 48 (follow-up) from HTX, in nondiabetic and diabetic patients. *P < 0.05 vs nondiabetics, §P < 0.05 vs basal values. B Levels of Ang-1–9, Ang 1–7, MAS1, and NFAM1 assessed 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%) *P < 0.05 vs diabetic patients in good glycemic control (< 7%), §P < 0.05 vs basal values

Fig. 7

A Fibrosis (green) in heart specimens from nondiabetic patient, diabetic patient with poor glycemic control (mean HbA1c 8.1 ± 0.4%), and diabetic patient with good glycemic control (mean HbA1c 6.6 ± 0.2%). B Percent values of fibrosis (ZEN 2.5 pro software) in explanted hearts (HTX) at week 1 (Basal) and week 48 (Follow-up) from HTX in nondiabetic and diabetic patients. P < 0.05 vs nondiabetics, §P < 0.05 vs basal values. C Percent values of fibrosis (ZEN 2.5 pro software) 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%) *P < 0.05 vs diabetic patients in good glycemic control (< 7%), §P < 0.05 vs basal values