Abstract
BackgroundHeart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM). The present study sought to determine how myocardial copper status and key copper-proteins might become impaired by diabetes, and how they respond to treatment with the Cu (II)-selective chelator triethylenetetramine (TETA) in DCM.MethodsExperiments were performed in Wistar rats with streptozotocin (STZ)-induced diabetes with or without TETA treatment. Cardiac function was analyzed in isolated-perfused working hearts, and myocardial total copper content measured by particle-induced x-ray emission spectroscopy (PIXE) coupled with Rutherford backscattering spectrometry (RBS). Quantitative expression (mRNA and protein) and/or activity of key proteins that mediate LV-tissue-copper binding and transport, were analyzed by combined RT-qPCR, western blotting, immunofluorescence microscopy, and enzyme activity assays. Statistical analysis was performed using Student’s t-tests or ANOVA and p-values of < 0.05 have been considered significant.ResultsLeft-ventricular (LV) copper levels and function were severely depressed in rats following 16-weeks’ diabetes, but both were unexpectedly normalized 8-weeks after treatment with TETA was instituted. Localized myocardial copper deficiency was accompanied by decreased expression and increased polymerization of the copper-responsive transition-metal-binding metallothionein proteins (MT1/MT2), consistent with impaired anti-oxidant defences and elevated susceptibility to pro-oxidant stress. Levels of the high-affinity copper transporter-1 (CTR1) were depressed in diabetes, consistent with impaired membrane copper uptake, and were not modified by TETA which, contrastingly, renormalized myocardial copper and increased levels and cell-membrane localization of the low-affinity copper transporter-2 (CTR2). Diabetes also lowered indexes of intracellular (IC) copper delivery via the copper chaperone for superoxide dismutase (CCS) to its target cuproenzyme, superoxide dismutase-1 (SOD1): this pathway was rectified by TETA treatment, which normalized SOD1 activity with consequent bolstering of anti-oxidant defenses. Furthermore, diabetes depressed levels of additional intracellular copper-transporting proteins, including antioxidant-protein-1 (ATOX1) and copper-transporting-ATPase-2 (ATP7B), whereas TETA elevated copper-transporting-ATPase-1 (ATP7A).ConclusionsMyocardial copper deficiency and defective cellular copper transport/trafficking are revealed as key molecular defects underlying LV impairment in diabetes, and TETA-mediated restoration of copper regulation provides a potential new class of therapeutic molecules for DCM.
Highlights
Heart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM)
LV-copper levels were fully restored to normal concentrations when TETA treatment was instituted after 8-weeks’ diabetes and maintained for a further 8 weeks (Figure 1A): this effect was accompanied by substantial improvements in cardiac function, including in cardiac output, and indexes of systolic and diastolic function (Table 1)
TETA treatment accentuated diabetes-evoked upregulation of copper transporter-2 (CTR2), whereas it did not modify diabeteselicited lowering of Ctr1 expression Immunoblotting for the high-affinity copper transporter, copper transporter-1 (CTR1), indicated the presence of dimeric (~45 kD) and trimeric (~75 kD) forms, both of which were diminished in diabetic LV myocardium as compared to control (Figure 1B and 1C)
Summary
Heart disease is the leading cause of death in diabetic patients, and defective copper metabolism may play important roles in the pathogenesis of diabetic cardiomyopathy (DCM). Altered myocellular [K+]IC and [Na+]IC may contribute to the impaired cardiac function and energy-inefficient metabolism in DCM [19,20]
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