Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats

Abstract

Left ventricular (LV) dysfunction is a common comorbidity in diabetic patients, although the molecular mechanisms underlying this cardiomyopathic feature are not completely understood. Aldehyde dehydrogenase 2 (ALDH2) has been considered a key cardioprotective enzyme susceptible to oxidative inactivation. We hypothesized that hyperglycemia-induced oxidative stress would influence ALDH2 activity, and ALDH2 inhibition would lead to cardiac functional alterations in diabetic rats. Diabetes was induced by intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin. Rats were divided randomly into four groups: control, untreated diabetic, diabetic treated with N-acetylcysteine (NAC) and diabetic treated with α-lipoic acid (α-LA). Cardiac contractile function, oxidative stress markers and reactive oxygen species (ROS) levels were assessed. ALDH2 activity and expression also were determined. The role of ALDH2 activity in change in hyperglycemia-induced mitochondrial membrane potential (Δψ) was tested in cultured neonatal cardiomyocytes. Myocardial MDA content and ROS were significantly higher in diabetic rats than in controls, whereas GSH content and Mn-SOD activity were decreased in diabetic rats. Compared with controls, diabetic rats exhibited significant reduction in LV ejection fraction and fractional shortening, accompanied by decreases in ALDH2 activity and expression. NAC and α-LA attenuated these changes. Mitochondrial Δψ was decreased greatly with hyperglycemia treatment, and high glucose combined with ALDH2 inhibition with daidzin further decreased Δψ. The ALDH2 activity can be regulated by oxidative stress in the diabetic rat heart. ALDH2 inhibition may be associated with LV reduced contractility, and mitochondrial impairment aggravated by ALDH2 inhibition might reflect an underlying mechanism which causes cardiac dysfunction in diabetic rats.

Figure 1

Left ventricular functional analysis with echocardiography. (A) Representative M-mode echocardiograms from control (Con), untreated diabetic (Dia), diabetic treated with NAC (NAC) and diabetic treated with α-LA (α-LA) rats. LVESD, left ventricular end systolic diameter; LVEDD, left ventricular end diastolic diameter. (B) The mean left ventricular ejection fraction (EF) from Con, Dia, NAC and α-LA rats. *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Data are presented as mean ± SEM (n = 6–8). (C) The mean left ventricular fractional shortening (FS) from Con, Dia, NAC and α-LA rats. *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Data are presented as mean ± SEM (n = 6–8).

Figure 2

Reactive oxygen species (ROS) production in isolated rat hearts from control (Con), untreated diabetic (Dia), diabetic treated with NAC (NAC) and diabetic treated with α-LA (α-LA) rats. Intracellular ROS levels were determined by measuring the fluorescence of DCF with FACS (excitation 488 nm, emission 530 nm). *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Data are presented as mean ± SEM of the geometric mean fluorescence (n = 6–8).

Figure 3

ALDH2 dehydrogenase activity analysis. (A) The effect of NAC and α-LA treatment on mitochondrial aldehyde dehydrogenase 2 (ALDH2) activity in isolated rat hearts from control (Con), untreated diabetic (Dia), diabetic treated with NAC (NAC) and diabetic treated with α-LA (α-LA) rats. ALDH2 activity was determined by measuring the conversion of propionaldehyde to its propionic acid product. *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Data are mean ± SEM (n=6–8). (B) The effect of dithiothreitol (DTT) on ALDH2 activity in isolated heart mitochondria from control or diabetic rats. *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Data are mean ± SEM (n=4). (C) Representative Western blot showing various cellular markers in total homogenate (Homo) and isolated mitochondria (Mito) prepared from rat hearts.

Figure 4

Correlation between myocardial ALDH2 activity and EF (A) and FS (B). Data were analyzed by linear regression. *P < 0.05.

Figure 5

(A) Western blots of ALDH2 expression from control (Con), untreated diabetic (Dia), diabetic treated with NAC (NAC) and diabetic treated with α-LA (α-LA) rats. β-actin was an internal control. *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Results are presented as mean ± SEM (n=6–8). (B) Representative immunohistochemistrical staining for 4-hydroxy-2-nonenal (HNE)-protein adducts from Con, Dia, NAC and α-LA rats. Scale bar, 50 μm. (C) Western blots of Mn-SOD protein expression from Con, Dia, NAC and α-LA rats. β-actin was an internal control. *P < 0.05 versus control group; **P < 0.05 versus diabetic group. Results are presented as mean ± SEM (n = 6–8).

Figure 6

The change in Δψ was quantified from normal glucose (LG), normal glucose+daidzin (LG+daidzin), high glucose (HG) and high glucose+daidzin (HG+daidzin) group. *P < 0.05 versus LG group; ^#P < 0.05 versus HG group. Data are presented as mean ± SEM (n = 4).