Advanced glycation endproduct (AGE) accumulation and AGE receptor (RAGE) up-regulation contribute to the onset of diabetic cardiomyopathy

Abstract

Diabetic cardiomyopathy is manifested by compromised systolic and diastolic function. This study was designed to examine the role of advanced glycation endproduct (AGE) and AGE receptor (RAGE) in diabetic cardiomyopathy. Heart function was assessed in isolated control and streptozotocin-induced diabetic hearts following in vivo RAGE gene knockdown using RNA interference. Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS) and time-to-90% relengthening (TR(90)). RAGE was assayed by RT-PCR and immunoblot. Diabetes significantly enhanced cardiac MG, AGE and RAGE levels accompanied with colocalization of AGE and RAGE in cardiomyocytes. Diabetes-elicited increase in RAGE was inhibited by in vivo siRNA interference. The AGE formation inhibitor benfotiamine significantly attenuated diabetes-induced elevation in MG, AGE, RAGE and collagen cross-linking without affecting hypertriglyceridaemia and hypercholesterolaemia in diabetes. Diabetes markedly decreased LV contractility, as evidenced by reduced +/-dP/dt and LV developed pressure (LVDP), which were protected by RAGE gene knockdown. In addition, MG-derived AGE (MG-AGE) up-regulated cardiac RAGE mRNA and triggered cardiomyocyte contractile dysfunction reminiscent of diabetic cardiomyopathy. The MG-AGE-elicited prolongation of TPS and TR(90) was ablated by an anti-RAGE antibody in cardiomyocytes. Interestingly, MG-AGE-induced cardiomyocyte dysfunction was associated with mitochondrial membrane potential (MMP) depolarization and reduced GSK-3beta inactivation in control cardiomyocytes, similar to those from in vivo diabetes. Treatment with siRNA-RAGE ablated diabetes-induced MMP depolarization and GSK-3beta inactivation. Collectively, our result implicated a role of AGE-RAGE in the pathogenesis of diabetic cardiomyopathy.

Figure 1

Effect of diabetes or RAGE knockdown on cardiac methylglyoxal levels and RAGE expression. (A, B) Representative HPLC chromatographic traces of methylglyoxal from control and diabetic hearts. Peak I and peak II depict the quinoxaline derivate of methylglyoxal‐2‐methylquinoxaline (2‐MQ) and the quinoxaline internal standard‐5‐methylquinoxaline (5‐MQ), respectively; (C) Pooled data of methylglyoxal levels; (D) RAGE expression in diabetes with or without 48‐hr siRNA silence. Inset: Representative gel blots of RAGE and β‐actin using specific antibodies. Mean ± S.E.M., n= 6–10, *P < 0.05 versus control group, ^# P < 0.05 versus diabetic group.

Figure 2

Effect of diabetes on cardiac AGE formation and AGE‐RAGE colocalization. (A, B) Representative immunofluorescent images depicting AGE distribution in control and diabetic ventricular tissues; (C, D) AGE‐RAGE colocalization in control mouse cardiomyocytes; (E, F) AGE‐RAGE colocalization in diabetic mouse cardiomyocytes. For panel C through F, myocytes were probed with an anti‐AGE antibody followed by a fluorescein isothiocyanate‐conjugated anti‐mouse IgG for AGE detection. The cover slips were then incubated with a rabbit anti‐RAGE antiserum and a rhodamine‐conjugated goat anti‐rabbit IgG for RAGE localization.

Figure 3

Effect of benfotiamine (BT, 80 mg/kg/day for 6 weeks) on (A) methylglyoxal, (B) RAGE expression, (C) AGE formation, (D) collagen cross‐linking, (E) plasma triglycerides and (F) plasma cholesterol levels in ventricular tissues or plasma from control and STZ‐induced diabetic mice. Mean ± S.E.M., n= 5, *P < 0.05 versus control group, ^# P < 0.05 versus diabetic group.

Figure 4

(A) RAGE mRNA level measured by RT‐PCR in control and diabetic cardiomyocytes treated with MG‐AGE (0–5 μmol/l) for 4 hrs; (B) peak shortening (PS) amplitude; (C) time‐to‐PS (TPS) and (D): time‐to‐90% relengthening (TR₉₀) in control and diabetic cardiomyocytes treated with siRNA‐RAGE in vivo or MG‐AGE (2.5 μmol/l for 4 hrs in vitro) in the absence or presence of an anti‐RAGE antibody to cancel the interaction between MG‐AGE and RAGE. Non‐immune IgG (NI‐IgG) was used as control. Mean ± S.E.M., n= 4 (panel A) or 25–36 (panel B–D) per group, *P < 0.05 versus control, ^# P < 0.05 versus diabetic, †P < 0.05 versus control + MG‐AGE group.

Figure 5

Cardiomyocyte mitochondrial membrane potential (MMP) from control cardiomyocytes incubated with BSA (A, 2.5 μmol/l), MG‐AGE (B, 2.5 μmol/l) or CCCP (C, 10 μmol/l) for 1 and 4 hrs as well as STZ‐induced diabetic myocytes (D, E) Kinetic changes of MMP in response to MG‐AGE or siRNA treatment using JC‐1 fluorochrome (ratio of red to green fluorescence); (F) MMP between control and diabetic mice with srRNA‐RAGE or siRNA‐NT treatment. Mean ± S.E.M., n= 4, *P < 0.05 versus BSA or control group, ^# P < 0.05 versus diabetic or MG‐AGE group.

Figure 6

Time‐dependent phosphorylation of GSK‐3 β (serine‐9) in response to MG‐AGE and control BSA (2.5 μmol/l). (A) GSK‐3β phosphorylation by BSA and MG‐AGE (0–4 hrs); (B) Pooled data of GSK‐3 β phosphorylation in control myocytes; (C) pooled data of GSK‐3β phosphorylation in control and diabetic hearts treated with siRNA‐RAGE or siRNA‐NT. Inset: Representative gels of GSK‐3β phosphorylation using specific antibodies. Mean ± S.E.M., n= 4, *P < 0.05 versus BSA or control group, ^# P < 0.05 versus MG‐AGE at time 0 or diabetic group.