Aldose reductase mediates myocardial ischemia-reperfusion injury in part by opening mitochondrial permeability transition pore

Abstract

Aldose reductase (AR), a member of the aldo-keto reductase family, has been demonstrated to play a central role in mediating myocardial ischemia-reperfusion (I/R) injury. Recently, using transgenic mice broadly overexpressing human AR (ARTg), we demonstrated that AR is an important component of myocardial I/R injury and that inhibition of this enzyme protects heart from I/R injury (20-22, 48, 49, 56). To rigorously delineate mechanisms by which AR pathway influences myocardial ischemic injury, we investigated the role played by reactive oxygen species (ROS), antioxidant enzymes, and mitochondrial permeability transition (MPT) pore opening in hearts from ARTg or littermates [wild type (WT)] subjected to I/R. MPT pore opening after I/R was determined using mitochondrial uptake of 2-deoxyglucose ratio, while H2O2 was measured as a key indicator of ROS. Myocardial 2-deoxyglucose uptake ratio and calcium-induced swelling were significantly greater in mitochondria from ARTg mice than in WT mice. Blockade of MPT pore with cyclosphorin A during I/R reduced ischemic injury significantly in ARTg mice hearts. H2O2 measurements indicated mitochondrial ROS generation after I/R was significantly greater in ARTg mitochondria than in WT mice hearts. Furthermore, the levels of antioxidant GSH were significantly reduced in ARTg mitochondria than in WT. Resveratrol treatment or pharmacological blockade of AR significantly reduced ROS generation and MPT pore opening in mitochondria of ARTg mice hearts exposed to I/R stress. This study demonstrates that MPT pore opening is a key event by which AR pathway mediates myocardial I/R injury, and that the MPT pore opening after I/R is triggered, in part, by increases in ROS generation in ARTg mice hearts. Therefore, inhibition of AR pathway protects mitochondria and hence may be a useful adjunct for salvaging ischemic myocardium.

Fig. 1.

Mitochondrial permeability in wild-type (WT), human aldose reductase expressing transgenic (ARTg), and aldose reductase inhibited ARTg (ARTg-ARI) (using zopolrestat) mice hearts subjected to ischemia-reperfusion (I/R; n = 6 per group). Mitochondrial permeability transition (MPT) pore opening was determined using 2-[³H]deoxyglucose (2-DG) uptake in the mitochondria of hearts subjected to baseline perfusion (A) or I/R conditions denoted as B/I/R (B) or swelling of mitochondria by measuring light scattering at 540 nm in the presence and absence of varying amounts of added calcium (μM) (n = 6 per group) (C). Data represent means ± SD. Details of the experimental conditions for data are described in methods. ΔOD, change in optical density.

Fig. 2.

Determination of myocardial ischemic injury, as shown by creatine kinase (CK) release (A), and left ventricular developed pressure (LVDP) recovery in WT, ARTg, cyclosporin A (CsA)-treated WT, and CsA-treated ARTg mice hearts subjected to I/R (B). Six hearts per group were studied. Data represent means ± SD. Details of the experimental conditions are described in methods.

Fig. 3.

GSH measurements in mitochondrial fractions (A) and total lysates (B) from ARTg, ARTg-ARI, WT, and WT-ARI mice hearts were determined as described in methods. Data represent means ± SD; n = 6–9 per group.

Fig. 4.

A: SOD activity at baseline or after I/R in ARTg and WT heart homogenates. B: malonaldialdehyde (MDA) was measured in heart lysates from WT, WT-ARI, ARTg, and ARTg-ARI after I/R injury. The activity levels were determined using commercially available kits from Oxis. Data represent means ± SD; n = 6 per group.

Fig. 5.

A: measurements of mitochondrial H₂O₂ after baseline perfusion in WT, WT-ARI, ARTg, and ARTg-ARI, mice hearts. B: H₂O₂ was measured in isolated mitochondria from the WT, WT-ARI, ARTg, and ARTg-ARI mice hearts after I/R. The details of the amplex red method to measure mitochondrial H₂O₂ are described in methods. Data represent means ± SD; n = 6 per group.

Fig. 6.

Effect of resveratrol (Res) on H₂O₂ generation by mitochondria under baseline (A) and I/R conditions, denoted as B/I/R (B), Ca²⁺-induced mitochondrial swelling (C), and ischemic injury (D) in WT and ARTg mice hearts subjected to I/R. Data represent means ± SD; n = 6 in each group. Details of the experimental conditions for data are described in methods.