VDAC: old protein with new roles in diabetes

Abstract

A decrease in capillary density due to an increase in endothelial cell apoptosis in the heart is implicated in cardiac ischemia in diabetes. The voltage-dependent anion channel (VDAC) plays a crucial role in the regulation of mitochondrial metabolic function and mitochondria-mediated apoptosis. This study is designed to examine the role of VDAC in coronary endothelial dysfunction in diabetes. Endothelial cells (ECs) were more apoptotic in diabetic left ventricle of diabetic mice and mouse coronary ECs (MCECs) isolated from diabetic mice exhibited significantly higher mitochondrial Ca(2+) concentration and VDAC protein levels than control MCECs. The expression of VDAC-short hairpin RNA (shRNA) not only decreased the resting mitochondrial Ca(2+) concentration but also attenuated mitochondrial Ca(2+) uptake in diabetic MCECs. Furthermore, the downregulation of VDAC in diabetic MCECs significantly decreased mitochondrial superoxide anion (O(2)(-)) production and the activity of the mitochondrial permeability transition pore (mPTP) opening (an indirect indicator of cell apoptosis) toward control levels. These data suggest that the increased VDAC level in diabetic MCECs is responsible for increased mitochondrial Ca(2+) concentration, mitochondrial O(2)(-) production, and mPTP opening activity. Normalizing VDAC protein level may help to decrease endothelial cell apoptosis, increase capillary density in the heart, and subsequently decrease the incidence of cardiac ischemia in diabetes.

Fig. 1.

Augmented endothelial cell (EC) apoptosis in the left ventricle (LV) and increased voltage-dependent anion channel (VDAC) protein level and increased mitochondrial Ca²⁺ concentration ([Ca²⁺]_mit) in mouse coronary ECs (MCECs) isolated from diabetic mice. A: columns show summarized data of the percentage of apoptotic ECs (the number of apoptotic ECs divided by total number of ECs). Control (Cont), n = 5; diabetic (Dia), n = 6. Data are means ± SE. *P < 0.05 vs. control. B: representative images showing [Ca²⁺]_mit in MCECs isolated from control and diabetic mice (left photomicrographs). Right columns show summarized [Ca²⁺]_mit data (Rhod-2-fluorescence intensity from the cell minus background intensity). Cont, n = 25; Dia, n = 38. Data are means ± SE. *P < 0.05 vs. control. Bar = 50 μm. C: Western blots showing VDAC and actin protein levels (left). Actin was used as a loading control. Right columns show VDAC protein levels normalized by actin. Cont, n = 6; Dia, n = 6. Data are means ± SE. *P < 0.05 vs. control.

Fig. 2.

Effect of VDAC inhibition by VDAC-short hairpin RNA (shRNA) adenovirus (Adv) (VDAC-shRNA) on mitochondrial [Ca²⁺]_mit in MCECs. A: mouse VDAC shRNA was designed and cloned into an adenoviral vector with a U6 promoter. B: infection of mouse ECs with VDAC-shRNA significantly decreases VDAC protein levels. Western blots showing VDAC and actin protein levels (left). Actin was used as a loading control. Right columns show VDAC protein level normalized to actin. ECs infected with control Adv (Cont-Adv), n = 2; ECs infected with VDAC-shRNA, n = 2. Data are means ± SE. *P < 0.05 vs. control. C and D: measurement of [Ca²⁺]_mit in MCECs at rest and after treatment with the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor, cyclopiazonic acid (CPA), in the absence of extracellular Ca²⁺. C: time course of the [Ca²⁺]_mit measurement experiments. Resting level indicates [Ca²⁺]_mit without treatment in the presence of Ca²⁺ in the extracellular media. After changing the extracellular media to Ca²⁺-free media, CPA (10 μmol/l) was added to test Ca²⁺ uptake by mitochondria. Area under the curve (AUC) and Δpeak were used to analyze Ca²⁺ uptake. D: summarized [Ca²⁺]_mit data. Control ECs infected with control Adv (Cont-EC, Cont-Adv), n = 16; diabetic ECs infected with control Adv (Dia-EC, Cont-Adv), n = 19; diabetic ECs infected with VDAC-shRNA (Dia-EC, VDAC-shRNA), n = 22. Data are means ± SE. *P < 0.05 vs. Cont-EC, Cont-Adv. #P < 0.05 vs. Dia-EC, Cont-Adv.

Fig. 3.

Effect of VDAC-shRNA on mitochondrial O₂⁻ concentration ([O₂⁻]_mit) in diabetic MCECs. A: representative images showing [O₂⁻]_mit in MCECs isolated from control and diabetic mice infected with Cont-Adv or VDAC-shRNA (left photomicrographs). Bar = 25 μm. B: summarized data of [O₂⁻]_mit. Cont-EC, Cont-Adv, n = 41; Dia-EC, Cont-Adv, n = 44; Dia-EC, shRNA-VDAC, n = 30. Data are means ± SE. *P < 0.05 vs. Cont-EC, Cont-Adv. #P < 0.05 vs. Dia-EC, Cont-Adv.

Fig. 4.

Activity of the mitochondrial permeability transition pore (mPTP) opening is significantly increased in diabetic MCECs, and VDAC-shRNA expression decreases the activity toward the control level. The fluorescence intensity of calcein-AM in mitochondria was measured to assess the activity of the mPTP opening. The decrease in calcein-AM intensity indicates the increase of the mPTP opening. Cont-EC and Cont-Adv, n = 108; Dia-EC and Cont-Adv, n = 133; Dia-EC and VDAC-shRNA, n = 98. Data are means ± SE. *P < 0.05 vs. Cont-EC, Cont-Adv. #P < 0.05 vs. Dia-EC, Cont-Adv.

Fig. 5.

Hexokinase II (HK2), but not hexokinase I (HK1), is significantly decreased in diabetic MCECs. Western blots showing HK1, HK2, and actin protein levels (left). Actin was used as a loading control. Right columns show HK1 and HK2 protein expression levels normalized by actin. Cont, n = 4; Dia, n = 4. Data are means ± SE. *P < 0.05 vs. control.