Red Blood Cells in Type 2 Diabetes Impair Cardiac Post-Ischemic Recovery Through an Arginase-Dependent Modulation of Nitric Oxide Synthase and Reactive Oxygen Species

Abstract

This study tested the hypothesis that red blood cell (RBC) arginase represents a potential therapeutic target in ischemia-reperfusion in type 2 diabetes. Post-ischemic cardiac recovery was impaired in hearts from db/db mice compared with wild-type hearts. RBCs from mice and patients with type 2 diabetes attenuated post-ischemic cardiac recovery of nondiabetic hearts. This impaired cardiac recovery was reversed by inhibition of RBCs arginase or nitric oxide synthase. The results suggest that RBCs from type 2 diabetics impair cardiac tolerance to ischemia-reperfusion via a pathway involving arginase activity and nitric oxide synthase-dependent oxidative stress.

Keywords: ABH, 2 (S)-amino-6-boronohexanoic acid; KH, Krebs-Henseleit; L-NAME, NG-nitro-L-arginine methyl ester; LVDP, left ventricular developed pressure; LVEDP, left ventricular end-diastolic pressure; NAC, N-acetylcysteine; NO, nitric oxide; NOS, nitric oxide synthase; RBC, red blood cell; ROS, reactive oxygen species; WT, wild type; arginase; dP/dt, the first derivative of left ventricular pressure; eNOS, endothelial nitric oxide synthase; iNOS, inducible isoform of nitric oxide synthase; nitric oxide synthase; nor-NOHA, Nω-hydroxy-nor-L-arginine; reactive oxygen species; red blood cells; type 2 diabetes.

Graphical abstract

Figure 1

Arginase Expression and Activity Are Increased in RBC From Mice With Type 2 Diabetes (A) Western blot of arginase 1 expression in red blood cells (RBCs) from wild type (WT) (n = 6) and db/db mice (n = 6). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control. (B) Arginase activity in RBCs from WT (n = 4) and db/db (n = 5) mice. (C) Arginase 1 expression in RBCs from WT mice (n = 4) following incubation with 5 or 25 mmol/l glucose. (D) Arginase activity in RBCs from WT mice (n = 8) following incubation with 5 or 25 mmol/l glucose. Arginase activity in WT RBCs treated with 5 mmol/l glucose is used as standard. Significant differences between groups are shown; *p < 0.05, and **p < 0.01.

Figure 2

ROS Production Is Increased in RBCs From Mice With Type 2 Diabetes and Is Regulated by Arginase and NOS (A) Levels of reactive oxygen species (ROS) in RBCs from WT (n = 5) and db/db (n = 5) mice determined using electron spin resonance. (B to G) ROS levels in RBCs from WT and db/db mice determined using flow cytometry, (B) under basal condition (WT n = 6 and db/db n = 6), (C) following incubation with vehicle or the ROS scavenger N-acetylcysteine (NAC) (1 mmol/l, WT n = 6 and db/db n = 6), (D) following incubation with vehicle or the arginase inhibitor 2 (S)-amino-6-boronohexanoic acid (ABH) (0.1 mmol/l, WT n = 7 and db/db n = 7), (E) following incubation with vehicle or the nonselective nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) (0.1 mmol/l, WT n = 8 and db/db n = 8), (F) following incubation with the inducible isoform of NOS inhibitor 1400W (0.1 mmol/l, WT n = 8 and db/db n = 8, using electron spin resonance), and (G) following incubation with vehicle or L-arginine (3 mmol/l, WT n = 5 and db/db n = 5). ROS generation in WT RBCs is used as standard in B to F. Significant differences are shown; *p < 0.05, **p < 0.01, and ***p < 0.001. Abbreviations as in Figure 1.

Figure 3

RBCs From db/db Mice Impair Cardiac Post-Ischemic Functional Recovery (A) Hearts from WT mice were given Krebs-Henseleit (KH) buffer (WT heart + KH, n = 5) or RBCs from WT mice (WT heart + WT RBCs, n = 5), and hearts from db/db mice were given KH buffer (db/db heart + KH, n = 4) or RBCs from db/db mice (db/db heart + db/db RBCs, n = 9). (B) Hearts from WT mice were given RBCs from either WT (n = 5) or db/db (n = 5) mice. (C) Hearts from db/db mice were given RBCs from either WT (n = 5) or db/db (n = 6) mice. (D) Infarct size in WT hearts given RBCs from WT or db/db mice. Post-ischemic left ventricular developed pressure (LVDP) is expressed as percentage recovery from the pre-ischemic level. Significant differences between groups in A to C were analyzed using 2-way analysis of variance including all time points, whereas Student's t-test was performed in D; *p < 0.05 and ***p < 0.001. Abbreviations as in Figure 1.

Figure 4

RBCs From db/db Mice Impair Cardiac Post-Ischemic Recovery via an Arginase-Dependent Pathway (A) Hearts from WT mice were given RBCs from db/db mice incubated with vehicle (n = 5), N^ω-hydroxy-nor-L-arginine (nor-NOHA) 1 mmol/l (n = 8) or 3 mmol/l (n = 4). (B) Hearts from WT mice were given RBCs from db/db mice incubated with vehicle (n = 5) or ABH (1 mmol/l, n = 4). (C) Hearts from db/db mice were given KH buffer with either vehicle (n = 8) or nor-NOHA (1 mmol/l, n = 5). (D) Hearts from WT mice were given RBCs from WT mice incubated with vehicle (n = 6), the arginase inhibitor nor-NOHA (1 mmol/l, n = 10), nor-NOHA (3 mmol/l, n = 8) or L-NAME (0.1 mmol/l) + nor-NOHA (1 mmol/l, n = 6). (E) Hearts from db/db mice were given RBCs from WT mice incubated with vehicle (n = 5), nor-NOHA (1 mmol/l, n = 5) or L-NAME (0.1 mmol/l) + nor-NOHA (1 mmol/l, n = 5). Post-ischemic LVDP is expressed as percentage recovery from the pre-ischemic level. Significant differences between treatments were analyzed using 2-way analysis of variance including all time points; *p < 0.05 and ***p < 0.001. Abbreviations as in Figures 1, 2, and 3.

Figure 5

Inhibition of NOS Reverses the Impaired Cardiac Post-Ischemic Recovery Induced by db/db RBCs (A) Hearts from WT mice were given RBCs from db/db mice incubated with vehicle (n = 5), the arginase inhibitor nor-NOHA (1 mmol/l, n = 8) or the NOS inhibitor L-NAME (0.1 mmol/l) + nor-NOHA (1 mmol/l, n = 5). (B) Hearts from WT mice were given RBCs from db/db mice incubated with vehicle (n = 5), nor-NOHA (3 mmol/l, n = 4), or L-NAME (0.1 mmol/l) + nor-NOHA (3 mmol/l, n = 6). (C) Hearts from WT mice were given RBCs from db/db mice incubated with vehicle (n = 5) or L-NAME (0.1 mmol/l, n = 5). (D) Hearts from WT mice were given RBCs from WT mice incubated with vehicle (n = 6) or L-NAME (0.1 mmol/l, n = 5). Post-ischemic LVDP is expressed as percentage recovery from the pre-ischemic level. Significant differences between treatments were analyzed using 2-way analysis of variance including all time points; ***p < 0.001. Abbreviations as in Figures 1, 2, 3, and 4.

Figure 6

Effects of Antioxidant on the Impaired Recovery of LVDP Induced by db/db RBCs (A) Hearts from WT mice were given RBCs from db/db mice treated with vehicle (n = 7) or NAC (n = 7) for 4 weeks. (B) Hearts from WT mice were given RBCs from WT mice treated with vehicle (n = 6) or NAC (n = 7) for 4 weeks. LVDP is expressed as percentage recovery from the pre-ischemic level. Significant differences between treatments were analyzed using 2-way analysis of variance including all time points; **p < 0.01. Abbreviations as in Figures 1, 2, and 3.

Figure 7

Arginase Activity and ROS Production Are Increased in RBCs From Patients With Type 2 Diabetes (A) Arginase activity in RBCs from healthy subjects (HS, n = 9) and patients with type 2 diabetes (DM) (n = 9). (B to G) ROS production determined by electron spin resonance in RBCs from HS and DM (B) under basal condition (HS n = 6 and DM n = 6), (C) following incubation with vehicle (Veh) or the ROS scavenger NAC (1 mmol/l, HS n = 6 and DM n = 6), (D) following incubation with vehicle or the arginase inhibitor nor-NOHA (0.1 mmol/l, HS n = 6 and DM n = 6), (E) following incubation with vehicle or the nonselective NOS inhibitor L-NAME (0.1 mmol/l, HS n = 6 and DM n = 6), (F) following incubation with vehicle or the iNOS inhibitor 1400W (0.1 mmol/l, HS n = 5 and DM n = 6), and (G) following incubation with vehicle or L-arginine (L-Arg) (3 mmol/l, HS n = 6 and DM n = 6). Significant differences from vehicle or WT RBCs are shown; *p < 0.05, **p < 0.01 and ***p < 0.001. Abbreviations as in Figures 1, 2, and 4.

Figure 8

RBCs From Patients With Type 2 Diabetes Impair Post-Ischemic Cardiac Function of Isolated Rat Hearts via an Arginase and NOS-Dependent Mechanism Rat hearts were given (A) RBCs from either HS (n = 9) or patients with DM (n = 13), (B) RBCs from patients with DM and incubated with vehicle (n = 13), nor-NOHA (1 mmol/l, n = 12), or L-NAME (0.1 mmol/l) + nor-NOHA (1 mmol/l, n = 6), (C) RBCs from DM and incubated with vehicle (n = 13) or L-NAME (0.1 mmol/l, n = 5), (D) RBCs from HS and incubated with vehicle (n = 9) or L-NAME (0.1 mmol/l, n = 9), (E) RBCs from HS and incubated with vehicle (n = 6) or 1400W (0.1 mmol/l, n = 5) or RBCs from DM and incubated with vehicle (n = 5) or 1400W (n = 5), or (F) RBCs from HS incubated with vehicle (n = 9), nor-NOHA (1 mmol/l, n = 9), or L-NAME (0.1 mmol/l) + nor-NOHA (1 mmol/l, n = 9). Significant differences between treatments were analyzed using 2-way analysis of variance including all time points; *p < 0.05 and ***p < 0.001. Abbreviations as in Figures 1, 2, 3, and 4 and 7.