Protection of cardiac mitochondria by overexpression of MnSOD reduces diabetic cardiomyopathy.

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.

Cardiomyocyte dysfunction in models of type 1 and type 2 diabetes.

Cardiomyopathy is a major cause of mortality for both type 1 and 2 diabetic patients. However, experimental analysis of diabetic cardiomyopathy has focused on type 1 diabetes and there are few reports on cardiomyocyte dysfunction in the widely used type 2 diabetic model, db/db. In the current study, we assessed function in isolated ventricular myocytes from type 1 diabetic OVE26 mice and from type 2 diabetic db/db mice. When compared with their respective control strains, both diabetic models showed significant impairment in contractility, as assessed by percent peak shortening, maximal rate of contraction, and maximal rate of relaxation. The calcium decay rate was also significantly reduced in both types of diabetes, but the decrement was much greater in OVE26 myocytes, approx 50% vs only 20% in db/db myocytes. To understand the basis for slow calcium decay in diabetic myocytes and to understand the molecular basis for the quantitative difference between calcium decay in OVE26 and db/db myocytes, we measured cardiac content of the SERCA2a calcium pump. SERCA2a was significantly decreased in OVE26 diabetic myocytes but not reduced at all in db/db myocytes. The reduction of SERCA2a in OVE26 myocytes was completely prevented by overexpression of the antioxidant protein metallothionein, confirming that oxidative stress is an important component of diabetic cardiomyopathy. The current results demonstrate that though contractility is impaired in individual myocytes of db/db hearts and deficits are similar to what is seen in a severe model of type 1 diabetes, impairment in calcium reuptake is less severe, probably as a result of maintenance of normal levels of SERCA2a.

Overexpression of hexokinase protects hypoxic and diabetic cardiomyocytes by increasing ATP generation.

Cardiac glucose metabolism is critical to hypoxic cardiac function and hypoxia is known to stimulate glucose metabolism. This increases generation of ATP when mitochondrial respiration is inhibited. In diabetes, cardiac glucose metabolism declines and this may contribute to diabetic cardiomyopathy. The first step in committing glucose to metabolism is glucose phosphorylation catalyzed by hexokinase. But the potential role of hexokinase in the hypoxic or diabetic heart is uncertain. This study is designed to assess the ability of hexo-kinase and elevated ATP to protect cardiomyocyte contractility from hypoxia and diabetes. We used cardiomyocytes from the transgenic mouse Mh, which has cardiac specific expression of yeast hexokinase, to investigate the importance of glucose phosphorylation in the myocyte response to hypoxia and diabetes. Cardiomyocytes were isolated from FVB control and Mh hearts to assess the effects of 2 h of hypoxia on myocyte contractility and ATP content. The protective effect of hexokinase on diabetes was assessed in myocytes from the OVE26 Type I diabetic mouse and in OVE26Mh diabetic mice that carry the hexokinase gene. Overexpression of hexokinase had no effect during aerobic culture, but during hypoxia, hexokinase improved ATP content by 44% and this restored contractility almost to normal levels. In myocytes from diabetic mice, tested under both aerobic and hypoxic conditions, the hexokinase gene significantly improved ATP content and this significantly improved contractility. These results demonstrate that elevating hexokinase activity can be beneficial to hypoxic or diabetic cardiomyocytes secondary to improving myocyte ATP levels.

Cardiomyocyte defects in diabetic models and protection with cardiac-targeted transgenes.

Diabetic cardiomyopathy is a common chronic complication leading to heightened risk of heart failure among diabetic patients. In this chapter, we describe the methods for maintenance and breeding of two diabetic animal models, OVE26 and Agouti mice, for type 1 and type 2 diabetes, respectively. To understand the pathological mechanism, antioxidants such as manganese superoxide dismutase are overexpressed specifically in hearts of diabetic mice. Methods utilized to produce cardiac-targeted transgenic mice are presented in this chapter. Diabetic cardiomyopathy is evaluated in control, diabetic and transgene-protected diabetic animals by measuring contractility of isolated cardiomyocytes. Preparation and contractile analysis of cardiac myocytes are described in detail. Diabetic cardiomyocytes exhibit impaired contractility as well as delayed relaxation, and cardiac-overexpressed antioxidant transgenes are shown to reverse this damage.

Development of late-stage diabetic nephropathy in OVE26 diabetic mice.

OVE26 mice are a transgenic model of severe early-onset 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 microg/24 h in OVE26 vs. 20 microg/24 h in controls). Albumin excretion rate increased progressively with age and exceeded 15,000 microg/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.

Catalase protects cardiomyocyte function in models of type 1 and type 2 diabetes.

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.

Metallothionein prevents diabetes-induced deficits in cardiomyocytes by inhibiting reactive oxygen species production.

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.