We previously reported damage and elevated biogenesis in cardiac mitochondria of a type 1 diabetic mouse model and proposed that mitochondria are one of the major targets of oxidative stress. In this study, we targeted overexpression of the mitochondrial antioxidant protein manganese superoxide dismutase (MnSOD) to the heart to protect cardiac mitochondria from oxidative damage. Transgenic hearts had a 10-to 20-fold increase in superoxide dismutase (SOD) activity, and the transgenic SOD was located in mitochondria. The transgene caused a twofold increase in cardiac catalase activity. MnSOD transgenic mice demonstrated normal cardiac morphology, contractility, and mitochondria, and their cardiomyocytes were protected from exogenous oxidants. Crossing MnSOD transgenic mice with our type 1 model tested the benefit of eliminating mitochondrial reactive oxygen species. Overexpression of MnSOD improved respiration and normalized mass in diabetic mitochondria. MnSOD also protected the morphology of diabetic hearts and completely normalized contractility in diabetic cardiomyocytes. These results showed that elevating MnSOD provided extensive protection to diabetic mitochondria and provided overall protection to the diabetic heart. Diabetes 55: 798 -805, 2006 C ardiac failure is a leading cause of death for diabetic patients. Accumulated evidence indicates that heart failure in diabetes is due at least in part to a specific cardiomyopathy, referred to as diabetic cardiomyopathy, which is distinct from coronary arteriosclerosis. This was first proposed by Rubler et al. (1) in 1972 based on postmortem findings of heart failure in diabetic patients free of coronary artery disease. This finding has been confirmed by others in many subsequent clinical studies (2,3).Excess reactive oxygen species (ROS) production has been widely implicated in both the onset of diabetes and many of its complications (4 -6). Mitochondria are known to continuously generate superoxide radical as a byproduct of electron transport. The significance of mitochondria-generated ROS in diabetes has been proposed by several laboratories (7-11). Brownlee's laboratory provided strong evidence that ROS from mitochondria activate pathological pathways that induce diabetic complications (8,12,13). The normalization of these changes in high glucosecultured endothelial cells by overexpression of manganese superoxide dismutase (MnSOD), uncoupling protein-1, or inhibitors of mitochondrial electron transport (8) suggests that mitochondrial respiration acts as a major source of oxidative stress in diabetes complications. However, the role of mitochondrial oxidative stress has not been confirmed in diabetic cardiomyopathy.In a previous study, we observed defects in structure and function of mitochondria from diabetic heart (14) and proposed that mitochondria-derived ROS play an important causal role in mitochondrial damage and compensatory biogenesis. To confirm this hypothesis, we designed and constructed a transgenic line overexpressing the mitochondrial antioxi...

Diabetic cardiomyopathy is a common complication leading to heightened risk of heart failure and death. In the present report, we performed proteomic analysis on total cardiac proteins from the OVE26 mouse model of type 1 diabetes to identify protein changes that may contribute to diabetic cardiomyopathy. This analysis revealed that a surprising high proportion (12 of 20) of the altered proteins that could be identified by mass spectrometry were of mitochondrial origin. All but one of these proteins were upregulated by diabetes. Quantitative RT-PCR, performed for two of these proteins, indicated that part of the upregulation was attributed to increased messenger RNA levels. Morphological study of diabetic hearts showed significantly increased mitochondrial area and number as well as focal regions with severe damage to mitochondria. Diabetic mitochondria also showed reduced respiratory control ratio (9.63 +/- 0.20 vs. 6.13 +/- 0.41, P < 0.0001), apparently due to reduced state 3 rate, and diminished GSH level (5.5 +/- 0.9 vs. 8.2 +/- 2.5 micromol/mg protein, P < 0.05), indicating impaired mitochondrial function and increased oxidative stress. Further examination revealed increased mitochondrial DNA (1.03 +/- 0.18 vs. 0.69 +/- 0.13 relative copy number, P < 0.001) and a tendency to higher protein yield in OVE26 cardiac mitochondria, as well as increased mRNA level for mitochondrial transcription factor A and two mitochondrial encoded proteins. Taken together, these results show that mitochondria are a primary target in the diabetic heart, probably due to oxidative stress, and that this damage coincides with and may stimulate mitochondrial biogenesis.

Many diabetic patients suffer from a cardiomyopathy that cannot be explained by poor coronary perfusion. Reactive oxygen species (ROS) have been proposed to contribute to this cardiomyopathy. Consistent with this we found evidence for induction of the antioxidant genes for catalase in diabetic OVE26 hearts. To determine whether increased antioxidant protection could reduce diabetic cardiomyopathy, we assessed cardiac morphology and contractility, Ca 2؉ handling, malondialdehyde (MDA)-modified proteins, and ROS levels in individual cardiomyocytes isolated from control hearts, OVE26 diabetic hearts, and diabetic hearts overexpressing the antioxidant protein catalase. Diabetic hearts showed damaged mitochondria and myofibrils, reduced myocyte contractility, slowed intracellular Ca 2؉ decay, and increased MDA-modified proteins compared with control myocytes. Overexpressing catalase preserved normal cardiac morphology, prevented the contractile defects, and reduced MDA protein modification but did not reverse the slowed Ca 2؉ decay induced by diabetes. Additionally, high glucose promoted significantly increased generation of ROS in diabetic cardiomyocytes. Chronic overexpression of catalase or acute in vitro treatment with rotenone, an inhibitor of mitochondrial complex I, or thenoyltrifluoroacetone, an inhibitor of mitochondrial complex II, eliminated excess ROS production in diabetic cardiomyocytes. The structural damage to diabetic mitochondria and the efficacy of mitochondrial inhibitors in reducing ROS suggest that mitochondria are a source of oxidative damage in diabetic cardiomyocytes. We also found that catalase overexpression protected cardiomyocyte contractility in the agouti model of type 2 diabetes. These data show that both type 1 and type 2 diabetes induce damage at the level of individual myocytes, and that this damage occurs through mechanisms utilizing ROS.

Many diabetic patients suffer from cardiomyopathy, even in the absence of vascular disease. This diabetic cardiomyopathy predisposes patients to heart failure and mortality from myocardial infarction. Evidence from animal models suggests that reactive oxygen species play an important role in the development of diabetic cardiomyopathy. Our laboratory previously developed a transgenic mouse model with targeted overexpression of the antioxidant protein metallothionein (MT) in the heart. In this study we used MT-transgenic mice to test whether an antioxidant protein can reduce cardiomyopathy in the OVE26 transgenic model of diabetes. OVE26 diabetic mice exhibited cardiomyopathy characterized by significantly altered mRNA expression, clear morphological abnormalities, and reduced contractility under ischemic conditions. Diabetic hearts appeared to be under oxidative stress because they had significantly elevated oxidized glutathione (GSSG). Diabetic mice with elevated cardiac MT (called OVE26MT mice) were obtained by crossing OVE26 transgenic mice with MT transgenic mice. Hyperglycemia in OVE26MT mice was indistinguishable from hyperglycemia in OVE26 mice. Despite this, the MT transgene significantly reduced cardiomyopathy in diabetic mice: OVE26MT hearts showed more normal levels of mRNA and GSSG. Typically, OVE26MT hearts were found to be morphologically normal, and elevated MT improved the impaired ischemic contractility seen in diabetic hearts. These results demonstrate that cardiomyocyte-specific expression of an antioxidant protein reduces damage to the diabetic heart. Diabetes 51:174 -181, 2002

SUMMARY Inflammatory activation of myeloid cells is accompanied by increased glycolysis, which is required for the surge in cytokine production. Although in vitro studies suggest that increased macrophage glucose metabolism is sufficient for cytokine induction, the pro-inflammatory effects of increased myeloid cell glucose flux in vivo and the impact on atherosclerosis, a major complication of diabetes, are unknown. We therefore tested the hypothesis that increased glucose uptake in myeloid cells stimulates cytokine production and atherosclerosis. Overexpression of the glucose transporter GLUT1 in myeloid cells caused increased glycolysis and flux through the pentose phosphate pathway, but did not induce cytokines. Moreover, myeloid cell-specific overexpression of GLUT1 in LDL receptor-deficient mice was ineffective in promoting atherosclerosis. Thus, increased glucose flux is insufficient for inflammatory myeloid cell activation and atherogenesis. If glucose promotes atherosclerosis by increasing cellular glucose flux, myeloid cells do not appear to be the key targets.