Overexpression of p53 due to excess protein O-GlcNAcylation is associated with coronary microvascular disease in type 2 diabetes

Abstract

Aims: We previously reported that increased protein O-GlcNAcylation in diabetic mice led to vascular rarefaction in the heart. In this study, we aimed to investigate whether and how coronary endothelial cell (EC) apoptosis is enhanced by protein O-GlcNAcylation and thus induces coronary microvascular disease (CMD) and subsequent cardiac dysfunction in diabetes. We hypothesize that excessive protein O-GlcNAcylation increases p53 that leads to CMD and reduced cardiac contractility.

Methods and results: We conducted in vivo functional experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic (T2D) model, and EC-specific O-GlcNAcase (OGA, an enzyme that catalyzes the removal of O-GlcNAc from proteins)-overexpressing TH mice, as well as in vitro experiments in isolated ECs from these mice. TH mice exhibited a significant increase in coronary EC apoptosis and reduction of coronary flow velocity reserve (CFVR), an assessment of coronary microvascular function, in comparison to wild-type mice. The decreased CFVR, due at least partially to EC apoptosis, was associated with decreased cardiac contractility in TH mice. Western blot experiments showed that p53 protein level was significantly higher in coronary ECs from TH mice and T2D patients than in control ECs. High glucose treatment also increased p53 protein level in control ECs. Furthermore, overexpression of OGA decreased protein O-GlcNAcylation and down-regulated p53 in coronary ECs, and conferred a protective effect on cardiac function in TH mice. Inhibition of p53 with pifithrin-α attenuated coronary EC apoptosis and restored CFVR and cardiac contractility in TH mice.

Conclusions: The data from this study indicate that inhibition of p53 or down-regulation of p53 by OGA overexpression attenuates coronary EC apoptosis and improves CFVR and cardiac function in diabetes. Lowering coronary endothelial p53 levels via OGA overexpression could be a potential therapeutic approach for CMD in diabetes.

Keywords: Apoptosis; Capillaries; Cardiovascular disease; Coronary blood flow; Coronary microcirculation.

Graphical Abstract

Figure 1

Characterization of Tallyho (TH) mice. (A) Oral glucose tolerance test (OGTT). Wild-type mice (Wt, n_mice = 7); TH mice (TH, n_mice = 7). (B) Coronary flow velocity reserve (CFVR). Wt, n_mice = 6; TH, n_mice = 5. (C) Representative photographs showing the capillary density and EC apoptosis. ECs were stained by BS-lectin-FITC (a marker of ECs) and apoptotic cells were detected by TUNEL staining (red). An arrow indicates co-stained cells (apoptotic ECs, orange). Bar = 50µm. (D) Averaged data showing the capillary density in Wt (n_mice = 5) and TH (n_mice = 5). (E) Summarized data of the percentage of apoptotic ECs (the number of apoptotic ECs divided by total number of ECs). N_mice = 5 for each group. (F) Representative images of hearts after I/R. The area at risk (AAR) is shown in non-blue (= white + red) and the infarcted area is shown in white. Five cross sections (each 1 mm thick) from the ligation site to the apex. Averaged data of infarcted area in the AAR (G) and the AAR in the ventricle (H). N_mice = 6 per group. (I) Protein O-GlcNAcylation in MCECs was detected by the anti-O-GlcNAc antibody (RL2). Arrow indicates a representative band of proteins showing increased O-GlcNAcylation. Actin was used as a loading control. (J) Summarized data of the intensity of the band indicated by the arrow (left) and total RL2 signals (right) (n_mice = 7 per group). Data are mean ± S.E. *P < 0.05 vs. Wt. Statistical comparison between time-response curves was made by two-way ANOVA with Bonferroni post hoc test. Unpaired Student’s t-test was used for comparisons of two experimental groups.

Figure 2

Characterization of Dia-THOGA mice. (A) Western blots showing OGA and Actin protein levels in MCECs (left panel). Right dot plot shows OGA protein level normalized to Actin. N_mice = 5 per group. (B) Protein O-GlcNAcylation in MCECs detected with RL2 antibody (left). Dot plots show the summarized data of the intensity of the band indicated by the arrow (A and B) and total RL2 signals (right). N_mice = 5 per group. (C) OGTT. Wt, n_mice = 7; Dia-THOGA w/o DOX, n_mice = 11; Dia-THOGA + DOX, n_mice = 9. (D) CFVR. Wt, n_mice = 6; Dia-THOGA w/o DOX, n_mice = 8; Dia-THOGA + DOX, n_mice = 6. (E) Capillary density. Wt, n_mice = 8; Dia-THOGA w/o DOX, n_mice = 6; Dia-THOGA + DOX, n_mice = 8. (F) Apoptotic ECs. Wt, n_mice = 8; Dia-THOGA w/o DOX, n_mice = 6; Dia-THOGA + DOX, n_mice = 8. Data are mean ± S.E. *P < 0.05 vs. Wt, ^#P < 0.05 vs. Dia-THOGA + DOX. Statistical comparison between time-response curves was made by two-way ANOVA with Bonferroni post hoc test. Statistical comparison between three groups was made by one-way ANOVA with Bonferroni post hoc test.

Figure 3

Effect of hyperglycaemia and OGA overexpression on protein O-GlcNAcylation, p53 protein expression, and tube formation. (A) Representative Western blot image of OGA, O-GlcNAcylation (RL2 antibody), and Actin. (B) OGA protein levels in HCECs treated with normal glucose (NG, 5 mmol/L glucose + 20 mmol/L Mannitol, 4 days) or high glucose (HG, 25 mmol/L glucose, 4 days) with control adenovirus (Cont-Adv) or OGA-Adv transduction. N_experiments = 6 per group. *P < 0.05 vs. NG + Cont-Adv. (C) Protein O-GlcNAcylation detected by RL2 antibody. N_experiments = 6 per group. *P < 0.05 vs. NG + Cont-Adv, ^#P < 0.05 vs. HG + Cont-Adv. (D) p53 protein levels detected by Western blot. N_experiments = 8 per group. *P < 0.05 vs. NG + Cont-Adv, ^#P < 0.05 vs. HG + Cont-Adv. (E) p53 protein levels in HCECs from control and diabetic patients detected by Western blot. Control patients (C, n_patients = 5); and T2D patients (D, n_patients = 4). *P < 0.05 vs. Cont. (F) p53 protein levels in MCECs freshly isolated from Wt and TH mice detected by Western blot. N_mice = 15 per group. *P < 0.05 vs. Wt. (G) p53 protein levels in MCECs freshly isolated from Wt, Dia-THOGA w/o DOX and Dia-THOGA + DOX detected by Western blot. N_mice = 6 per group. *P < 0.05 vs. Wt. (H) Representative photograph image of tube formation. (I) Summarized data of total length, branch numbers, and junction numbers in control HCECs with vehicle (0.008% DMSO), diabetic HCECs with vehicle, and diabetic HCECs with PFT treatment (20 µmol/L, 24 h). N_experiments = 6 per group. *P < 0.05 vs. Cont + vehicle, ^#P < 0.05 vs. Dia + vehicle. Data are mean ± S.E. Unpaired Student’s t-test was used for comparisons of two experimental groups. Statistical comparison between three groups was made by one-way ANOVA with Bonferroni post hoc test.

Figure 4

Effect of chronic PFT treatment in TH mice on haemodynamics and mRNA levels of genes involved with p53 signalling. (A) OGTT. Wt + vehicle, n_mice = 10; TH + vehicle, n_mice = 11; TH + PFT, n_mice = 10. (B) CFVR. Wt + vehicle, n_mice = 8; TH + vehicle, n_mice = 9; TH + PFT, n_mice = 9. (C) Capillary density. Wt + vehicle, n_mice = 5; TH + vehicle, n_mice = 5; TH + PFT, n_mice = 6. (D) Apoptotic ECs. Wt + vehicle, n_mice = 5; TH + vehicle, n_mice = 5; TH + PFT, n_mice = 6. (E and F) Infarcted area in the AAR (E) and the AAR in the ventricle (F). N_mice = 6 per group. (G) p53 mRNA levels in MCECs. Wt + vehicle, n_mice = 8; TH + vehicle, n_mice = 8; TH + PFT, n_mice = 8. (H) Tnf mRNA levels in MCECs. Wt + vehicle, n_mice = 7; TH + vehicle, n_mice = 6; TH + PFT, n_mice = 6. (I) Bax mRNA levels in MCECs. Wt + vehicle, n_mice = 7; TH + vehicle, n_mice = 8; TH + PFT, n_mice = 7. (J) Bbc3 mRNA levels in MCECs. Wt + vehicle, n_mice = 7; TH + vehicle, n_mice = 7; TH + PFT, n_mice = 7. (K) Casp2 mRNA levels in MCECs. Wt + vehicle, n_mice = 7; TH + vehicle, n_mice = 6; TH + PFT, n_mice = 6. Data are mean ± S.E. *P < 0.05 vs. Wt + vehicle, ^#P < 0.05 vs. TH + vehicle. Statistical comparison between time-response curves was made by two-way ANOVA with Bonferroni post hoc test. Statistical comparison between three groups was made by one-way ANOVA with Bonferroni post hoc test.