Oxidative stress and diabetic cardiomyopathy

Diabetic cardiomyopathy, a heart muscle disease independent of micro-/macro-vascular complications, is manifested by prolonged action potential duration and prolonged cardiac contraction and relaxation. Several rationales have been postulated for the pathogenesis of diabetic cardiomyopathy including reactive oxygen species generation, although the precise mechanism is still undefined. My central hypothesis is that oxidative damage is the ultimate cause of diabetic cardiomyopathy. In vivo: Mice with cardiac-specific overexpression of the antioxidant metallothionein (MT) were made diabetic with the β-cell toxin streptozotocin (STZ) and isolated myocytes were studied. In vitro: Doxorubicin (Dox), a potent anti-cancer drug and reactive oxygen species donor and STZ were administered to myocytes isolated from adult rats. Vitamin C (100 μM) and the p38 MAP kinase inhibitor SB203580 (10 μM) were administered with either Dox or STZ. Contractile parameters, including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR ₉₀) and maximal velocities of shortening (+dL/dt) and relengthening (−dL/dt), were evaluated in mouse and rat myocytes. The calcium-sensitive fluorescent dye fura-2AM was used to evaluate intracellular Ca2+. Tissue oxidative stress and apoptosis were assessed with reduced/oxidized glutathione (GSH/GSSG) and caspase-3 assay, respectively. Western blotting was used to determine the expression of sarco(endo)plasmic reticulum Ca 2+-ATPase, Na+/Ca2+ exchanger, phospholamban, NADPH oxidase and angiotensin II receptors.; Myocytes treated with STZ and Dox both showed concentration-dependent inhibitions in myocyte contraction, which were partially preventable by pretreatment with either vitamin C or SB203580. STZ also depressed calcium-induced calcium release and slowed the fluorescence decay, whereas Dox had no effect on any parameters of intracellular Ca2+ clearing. Myocyte dysfunction evident in the FVB diabetic mice was preventable by the MT transgene. Reactive oxygen species were elevated by in vitro STZ and Dox treatment and in the diabetic myocytes, which was also prevented by MT overexpression. GSH/GSSG ratio and caspase-3 activity were significantly elevated in diabetic myocytes, which were attenuated by MT. MT overexpression was shown to prevent the STZ-induced increase in PLB and p47phox NADPH oxidase. These data show that reactive oxygen species generation directly leads to cardiac dysfunction under normal or diabetic conditions, which is prevented by MT, suggesting the value of antioxidant therapy in diabetes.