Regeneration of the Heart in Diabetes by Selective Copper Chelation

Garth J.S. Cooper Anthony R.J. Phillips Soon Y. Choong Bridget L. Leonard David J. Crossman Dianne H. Brunton 'Etuate L. Saafi Ajith M. Dissanayake Brett R. Cowan Alistair A. Young Christopher J. Occleshaw Yih-Kai Chan Fiona E. Leahy Geraldine F. Keogh Gregory D. Gamble Grant R. Allen Ade` le J. Pope Peter D.W. Boyd Sally D. Poppitt Thomas K. Borg Robert N. Doughty John R. Baker Heart disease is the major cause of death in diabetes, a disorder characterized by chronic hyperglycemia and cardiovascular complications. Although altered systemic regulation of transition metals in diabetes has been the subject of previous investigation, it is not known whether changed transition metal metabolism results in heart disease in common forms of diabetes and whether metal chelation can reverse the condition. We found that administration of the Cu-selective transition metal chelator trientine to rats with streptozotocin-induced diabetes caused increased urinary Cu excretion compared with matched controls. A CuII-trientine complex was demonstrated in the urine of treated rats. In diabetic animals with established heart failure, we show here for the first time that 7 weeks of oral trientine therapy significantly alleviated heart failure without lowering blood glucose, substantially improved cardiomyocyte structure, and reversed elevations in left ventricular collagen and 1 integrin. Oral trientine treatment also caused elevated Cu excretion in humans with type 2 diabetes, in whom 6 months of treatment caused elevated left ventricular mass to decline significantly toward normal. These data implicate accumulation of elevated loosely bound Cu in the mechanism of cardiac damage in diabetes and support the use of selective Cu chelation in the treatment of this condition. Diabetes 53:2501-2508, 2004 - D(LVH), LV dysfunction, and coronary artery iabetes is accompanied by increased prevalence of left ventricular (LV) hypertrophy disease (1), but the mechanism by which hyperglycemia or associated metabolic abnormalities lead to or cause heart disease has remained obscure. Four main processes have been implicated in glucose-mediated vascular disease, and it has been suggested that the primary fault that leads to tissue damage is hyperglycemia-driven overproduction of superoxide by the mitochondrial electron-transport chain in endothelial cells (2). Normalizing mitochondrial superoxide production in endothelial cells in vitro was reported to block four pathways of hyperglycemic tissue damage (3). A number of therapeutic studies that used antioxidant or carbonyl-trapping agents have had variable outcomes, indicating that the pathways that lead to in vivo tissue damage may be incompletely understood (4). The biology of transition metals, such as Zn, Mn, Mo, Cr, V, Fe, and Cu, has previously been evaluated in the context of diabetes. In hemochromatosis, excess myocardial Fe can cause heart disease, and Fe-mediated islet damage results in diabetes (5). Myocardial Fe excess also causes heart disease in hemosiderosis, in association with excessive dietary Fe intake, or ineffective erythropoiesis as in thalassemia or sideroblastic anemia (6,7). Altered cardiac Fe metabolism is implicated in the mechanism of heart disease in additional circumstances (8). Defective Cu metabolism is said to impair cardiovascular health in at least two known settings, chronic Cu deficiency (9,10) and defective intracellular Cu transport to mitochondrial cytochrome C oxidase caused by missense mutations in the second cytochrome c oxidase assembly gene, SCO2 (11). Thus, Cu deficiency has been implicated as a defect of Cu homeostasis that can lead to cardiac disease (12). By contrast, heart disease is not noted in chronic intracellular Cu overload, e.g., Wilsons disease (13). Free Fe and Cu ions are the most redox-active in mammalian tissues (14), where they may contribute to tissue damage by generation of reactive oxygen species such as hydroxyl radicals (15). However, the in vivo availability of catalytic Fe and Cu is usually very restricted, which serves as an important antioxidant defense (14). Whether Fe- or Cu-catalyzed redox reactions may play some role in diabetes complications has been discussed previously (4,16). It has not been established that such mechanisms or, for example, altered transition metal metabolism play a role in the forms of heart disease that complicate the major classes of diabetes, type 1 and type 2. Previously, administration of an Fe chelator and a Cu chelator in a 2-week experiment in diabetic rats was reported to ameliorate decreases in sciatic motor nerve conduction velocity, restore nutritive endoneurial blood flow, decrease systemic arterial pressure, and cause supranormal sciatic nutritive vascular conductance (17). We used trientine to probe relationships between Fe and Cu and diabetic heart disease, and the effects of Cu removal in diabetic rats and in people with diabetes. Trientine binds CuII selectively (log Kf 20) but also has significant affinity for ZnII (log Kf 12), FeII (log Kf 8), and FeIII (18). It is used clinically in the therapy of Wilsons disease, an inherited Cu-transporter defect that causes Cu accumulation and localized organ damage (19,20). We show here that trientine causes increased urinary Cu output in diabetic rats and humans compared with treated controls. Trientine reversed heart failure in diabetic rats, and diabetic rats and humans demonstrate improved cardiac structure after chronic trientine treatment. Potential mechanisms include regeneration of f-actin and normalization of collagen. Excess loosely bound Cu thus is implicated in the mechanism by which diabetes damages the heart. RESEARCH DESIGN AND METHODS Reagents and ethical and regulatory approvals. All reagents were from Sigma unless otherwise stated. All studies were approved by relevant ethics and regulatory committees. Urinary metal excretion in rats. Male Wistar rats (303 3 g) received an injection of streptozotocin (STZ; 55 mg/kg i.v.) or saline. Diabetes was diagnosed as blood glucose 11 mmol/l and insulin was not replaced. After 7 weeks of diabetes, rats were anesthetized, ureters were catheterized, and urine was collected. Animals were ventilated, and end-tidal CO2 and body temperature were maintained at 35 40 mmHg and 37C, respectively, with saline replacement of fluid loss. Trientine (triethylenetetramine dihydrochloride; Fluka) was infused (intravenously, 60 s once hourly) in increasing doses (0.1, 1.0, 10, and 100 mg/kg in 75 l of physiological saline), whereas controls received saline alone. Urine (15-min aliquots) was centrifuged, and supernatants were diluted (1:25 [vol/vol]) in 0.02 mol/l HNO3. Metals were determined by graphite furnace-atomic absorption spectrophotometry (Perkin Elmer), and X-band electron paramagnetic resonance (EPR) spectra were obtained (77K, Varian E3). Cardiac function in rats. After 6 weeks of diabetes, rats were assigned to one of four groups: untreat (...truncated)