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...

OVE26 mice are a transgenic model of severe earlyonset type 1 diabetes. These mice develop diabetes within the first weeks of life and can survive well over a year with no insulin treatment, and they maintain near normal body weight. To determine whether OVE26 mice provide a valuable model of chronic diabetic nephropathy (DN), OVE26 diabetic mice were compared with their nondiabetic littermates for functional and structural characteristics of DN. OVE26 mice exhibited pronounced polyuria and significant albuminuria by 2 months of age (305 g/24 h in OVE26 vs. 20 g/24 h in controls). Albumin excretion rate increased progressively with age and exceeded 15,000 g/24 h at 9 months of age. The profound loss of albumin led to hypoalbuminemia in some diabetic animals. Albuminuria coincided with an elevation in blood pressure as measured by tail cuff. The glomerular filtration rate (GFR) in OVE26 mice measured using fluorescein isothiocynate inulin clearance demonstrated that GFR increased significantly from 2 to 3 months of age and then decreased significantly from 5 to 9 months. GFR in 9-month-old diabetic mice was significantly lower than that of 9-month-old control mice. The decline in GFR coincided with a significant increase in renal vascular resistance. Structural studies showed an almost twofold increase in kidney weight between 2 and 5 months. Diabetic mice also showed progressively enlarged glomeruli and expanded mesangium with diffuse and nodular expansion of mesangial matrix. Tubulointerstitial fibrosis was also observed in these mice. Glomerular basement membrane was thickened in OVE26 mice. In summary, OVE26 mice demonstrate that most of the characteristics of human DN can be produced by chronic hyperglycemia in a murine model. This model will be useful for improved understanding and treatment of DN. A variety of experimentally induced or spontaneously hyperglycemic animals are used as models of human diabetes, such as streptoztocin-induced diabetic rats, NOD mice, and db/db mice. The kidney disease in many of these animals has been characterized (2,3), but none display the full array of features characteristic of human DN. In fact, the current mouse models primarily display features consistent with the earliest phase of DN, such as microalbuminuria (4,5). This is not surprising since these mouse models typically suffer from diabetes for several months, while the complete pattern of human DN requires decades to develop.In the current article, we follow the development of DN in a transgenic model of insulinopenic diabetes, the OVE26 mouse (6). The advantages of this model for the study of complications are straightforward: direct damage is limited to the ␤-cell, diabetes develops early, and very severe diabetes lasts for Ͼ1 year. Our results show that with respect to albuminuria, mesangial matrix accumulation, glomerular filtration rate (GFR), and interstitial fibrosis, OVE26 mice are significantly closer to advanced human DN than other available mouse models. RESARCH DESIGN AND METHODSOVE26 mice on the FVB ba...

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.

SC. Hydrogen sulfide ameliorates hyperhomocysteinemia-associated chronic renal failure. Am J Physiol

Many individuals with diabetes experience impaired cardiac contractility that cannot be explained by hypertension and atherosclerosis. This cardiomyopathy may be due to either organ-based damage, such as fibrosis, or to direct damage to cardiomyocytes. Reactive oxygen species (ROS) have been proposed to contribute to such damage. To address these hypotheses, we examined contractility, Ca 2؉ handling, and ROS levels in individual cardiomyocytes isolated from control hearts, diabetic OVE26 hearts, and diabetic hearts overexpressing antioxidant protein metallothionein (MT). Our data showed that diabetic myocytes exhibited significantly reduced peak shortening, prolonged duration of shortening/relengthening, and decreased maximal velocities of shortening/relengthening as well as slowed intracellular Ca 2؉ decay compared with control myocytes. Overexpressing MT prevented these defects induced by diabetes. In addition, high glucose and angiotensin II promoted significantly increased generation of ROS in diabetic cardiomyocytes. Chronic overexpression of MT or acute in vitro treatment with the flavoprotein inhibitor diphenyleneiodonium or the angiotensin II type I receptor antagonist losartan eliminated excess ROS production in diabetic cardiomyocytes. These data show that diabetes induces damage at the level of individual myocyte. Damage can be attributed to ROS production, and diabetes increases ROS production via angiotensin II and flavoprotein enzyme-dependent pathways.

BackgroundWe have previously reported the role of anti-angiogenic factors in inducing the transition from compensatory cardiac hypertrophy to heart failure and the significance of MMP-9 and TIMP-3 in promoting this process during pressure overload hemodynamic stress. Several studies reported the evidence of cardiac autophagy, involving removal of cellular organelles like mitochondria (mitophagy), peroxisomes etc., in the pathogenesis of heart failure. However, little is known regarding the therapeutic role of mitochondrial division inhibitor (Mdivi) in the pressure overload induced heart failure. We hypothesize that treatment with mitochondrial division inhibitor (Mdivi) inhibits abnormal mitophagy in a pressure overload heart and thus ameliorates heart failure condition.Materials and MethodsTo verify this, ascending aortic banding was done in wild type mice to create pressure overload induced heart failure and then treated with Mdivi and compared with vehicle treated controls.ResultsExpression of MMP-2, vascular endothelial growth factor, CD31, was increased, while expression of anti angiogenic factors like endostatin and angiostatin along with MMP-9, TIMP-3 was reduced in Mdivi treated AB 8 weeks mice compared to vehicle treated controls. Expression of mitophagy markers like LC3 and p62 was decreased in Mdivi treated mice compared to controls. Cardiac functional status assessed by echocardiography showed improvement and there is also a decrease in the deposition of fibrosis in Mdivi treated mice compared to controls.ConclusionAbove results suggest that Mdivi inhibits the abnormal cardiac mitophagy response during sustained pressure overload stress and propose the novel therapeutic role of Mdivi in ameliorating heart failure.

Previous studies in several strains of rats have demonstrated that 35 consecutive days of recurrent episodic hypoxia (7 h/day) cause an 8- to 13-mmHg persistent increase in diurnal systemic blood pressure (BP). Carotid chemoreceptors and the sympathetic nervous system have been shown to be necessary for development of this BP increase. The present study was undertaken to further define the role of renal artery sympathetic nerves and the adrenal medulla in this BP increase. Male Sprague-Dawley rats had either adrenal medullectomy, bilateral renal artery denervation, or sham surgery. Rats from each of these groups were subjected to episodic hypoxia for 35 days. Control groups received either compressed air or were left unhandled. Adrenal demedullation or renal artery denervation eliminated the chronic diurnal mean BP response (measured intra-arterially) to episodic hypoxia, whereas sham-operated controls continued to showed persistent elevation of systemic BP. Plasma and renal tissue catecholamine levels at the end of the experiment confirmed successful adrenal demedullation or renal denervation in the respective animals. The chronic episodic hypoxia-mediated increase in diurnal BP requires both intact renal artery nerves as well as an intact adrenal medulla.

WE, Tseng MT, Tyagi SC. Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia. myocyte N-methyl-D-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (ϪdL/dt), and maximal rate of relaxation (ϩdL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, ϪdL/dt, and ϩdL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1. myocyte; calcium; mitochondrial permeability; N-methyl-D-aspartate receptor-1; arrhythmogenesis THE PATHOPHYSIOLOGY of chronic heart failure (CHF) involves abnormalities in systolic and/or diastolic function and increases the propensity for reentry arrhythmias (30, 6). Continued elevation of cardiac sympathetic drive contributes to myocardial toxicity, leading to the decline in cardiac contractility (29). Recent observations suggest an increase in glutamatergic activity on sympathetic regulation, due to the upregulation of hypothalamic N-methyl-D-aspartate receptor-1 subunits (NMDA-R1) during CHF (16). Ischemia-and reperfusion-induced arrhythmias are sensitive to NMDA-R1 blockade (8).Hyperhomocysteinemia (HHCY) is a graded risk factor for CHF (12, 7) and for sudden cardiac death (SCD) resulting from coronary fibrous plaques (4, 1, 5). Homocysteinemia (HCY) induces interstitial cardiac fibrosis leading to systolic/diastolic dysfunction (13). The antagonist to the NMDA-R protects against HCY-induced oxidative damage in neurons (10) and protects against...