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Cardiac cell death in early diabetes : modulation by glutathione and polyunsaturated fatty acids Ghosh, Sanjoy

Abstract

Diabetes is a growing problem in Canada. Cardiovascular disease (CVD) remains the major cause of morbidity and mortality among diabetic patients. Although the majority of CVD are attributed to coronary vascular disease, an increasing body of evidence supports the existence of an additional heart muscle defect, in the absence of any vascular pathology. Among other factors, this heart disease is associated with excessive cell death in both human and animal models of diabetes. It is possible that this loss of cells progressively leads to interstitial fibrosis, myocardial hypertrophy and eventual loss of contractile function. Previous studies had established that oxidative stress due to excessive reactive oxygen species (ROS) generation and/or inadequate ROS neutralization, may be a key player in causing cardiac cell death via apoptosis in early diabetes. We used streptozotocin (STZ, 55 mg/kg) to make Wistar rats acutely diabetic and after 4 days examined the different processes that regulate cardiac glutathione (GSH), an important endogenous antioxidant. Reduction in myocyte GSH in diabetic rats was accompanied by increased oxidative stress, excessive reactive oxygen species, and an elevated apoptotic cell death. In this model, loss of GSH was not associated with any change in either biosynthesis or recycling of GSH, but with increased efflux of GSH out of diabetic cardiomyocytes. Gene expression of multidrug resistance protein 1, a transporter implicated in effluxing GSH during oxidative stress, was also elevated in these hearts. In the next study, we reported a loss of GSH specifically from the cardiac mitochondria (mitochondrial GSH, mGSH), the chief source of ROS. Such loss in mGSH could explain increased mitochondrial oxidative damage, and apoptosis via increases in caspase-9 and 3 activities in these hearts. Finally, the central role for mGSH loss in cardiac apoptosis was validated as exogenous GSH supplementation induced a robust amplification in mGSH and prevented apoptosis in diabetic rat hearts. Apart from antioxidant levels, changes in cellular lipid homeostasis have also been linked to cell death in early diabetes. However, most of the existing studies on the cytotoxicity of fatty acids are on vascular cell systems and with saturated fatty acids like palmitic acid. In the last few decades, saturated fats from the human diet have been replaced by ω-6 PUFA in form of vegetable oils. Thus, the effect of the more relevant ω-6 PUFA on the heart, especially during diabetes remains unknown. Male Wistar rats were first fed a diet rich in ω-6 PUFA [20% (wt/wt) sunflower oil] for 4 wk followed by streptozotocin to induce diabetes. After a brief period of hyperglycemia (4 days), hearts were excised for functional, morphological, and biochemical analysis. Interestingly, in diabetic rats, ω-6 PUFA decreased caspase-3 activity, crucial for myocardial apoptosis. However, cardiac necrosis, an alternative mode of cell death, increased. Interestingly, depletion of cardiac ATP stores could explain this "switch" to necrotic cell death in these animals. This was supported by mitochondrial abnormalities, impaired substrate utilization, and enhanced triglyceride accumulation in ω-6 PUFA fed diabetic hearts. Further analysis revealed that even in the absence of diabetes, a pro-inflammatory response is initiated in the heart following ω-6 PUFA feeding via augmented phospholipase A₂ activity and breakdown of cardiolipin, a mitochondrial phospholipid. ω-6 PUFA fed hearts also demonstrated elevated inducible nitric oxide synthase expression, loss of Mn superoxide dismutase, and increased mitochondrial nitrotyrosine levels. In these hearts, oxidative damage to mitochondrial DNA (mDNA) was demonstrated by 8-hydroxyguanosine immunopositivity, overexpression of DNA repair enzymes, and a decrease in the mRNA expression of specific respiratory subunits encoded by the mDNA. Thus, although promoted as being beneficial, excess dietary ω-6 PUFA, with its predisposition to cause mitochondrial nitrosative damage and necrosis following hyperglycemia, could accelerate myocardial abnormalities in diabetic patients.

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