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 Cu II -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.

Most patients with diabetes die from cardiac or arterial disease, for which there are limited therapeutic options. Free Cu 2ϩ ions are strongly pro-oxidant, and chelatable-Cu II is increased in the diabetic heart. We reported previously that treatment by Cu IIselective chelation with triethylenetetramine (TETA) evokes elevated urinary Cu II in diabetic rats and humans in whom it also improved hallmarks of established left ventricular (LV) disease. Here, we treated diabetic rats with TETA and evaluated its ability to ameliorate Cu 2ϩ -mediated LV and arterial damage by modifying the expression of molecular targets that included transforming growth factor (TGF)-␤1, Smad4, extracellular matrix (ECM) proteins, extracellular superoxide dismutase (EC-SOD), and heparan sulfate (HS). Eight-weeks of TETA treatment significantly improved cardiac diastolic function but not [glucose] plasma in diabetic animals. LV and aortic mRNAs corresponding to TGF-␤1, Smad4, collagen types I, III, and IV, and fibronectin-1, and plasminogen activator inhibitor-1, were elevated in untreated diabetic animals and normalized after TETA treatment. EC-SOD mRNA and protein, and [HS] tissue were significantly decreased in diabetes and restored by drug treatment. Candidate molecular mechanisms by which TETA could ameliorate diabetic cardiac and arteriovascular disease include the suppression of an activated TGF-␤/Smad signaling pathway that mediates increased ECM gene expression and restoration of normal EC-SOD and HS regulation. These findings are relevant to the restoration toward normal by TETA treatment of cardiac and arterial structure and function in diabetes.

Hypertension now affects about 600 million people worldwide and is a leading cause of death in the Western world. The spontaneously hypertensive rat (SHR), provides a useful model to investigate hypertensive heart failure (HF). The SHR model replicates the clinical progression of hypertension in humans, wherein early development of hypertension is followed by a long stable period of compensated cardiac hypertrophy that slowly progresses to HF. Although the hypertensive failing heart generally shows increased substrate preference towards glucose and impaired mitochondrial function, the cause-and-effect relationship between these characteristics is incompletely understood. To explore these pathogenic processes, we compared cardiac mitochondrial proteomes of 20-month-old SHR and Wistar-Kyoto controls by iTRAQ-labelling combined with multidimensional LC/MS/MS. Of 137 high-scoring proteins identified, 79 differed between groups. Changes were apparent in several metabolic pathways, chaperone and antioxidant systems, and multiple subunits of the oxidative phosphorylation complexes were increased (complexes I, III and IV) or decreased (complexes II and V) in SHR heart mitochondria. Respiration assays on skinned fibres and isolated mitochondria showed markedly lower respiratory capacity on succinate. Enzyme activity assays often also showed mismatches between increased protein expression and activities suggesting elevated protein expression may be compensatory in the face of pathological stress.

BackgroundDefective copper regulation is implicated as a causative mechanism of organ damage in diabetes. Treatment with trientine, a divalent-copper-selective chelator, improves arterial and renal structure/function in diabetes, wherein it also ameliorates left-ventricular (LV) hypertrophy. However, direct in vivo evidence that trientine can improve cardiac function in heart failure has hitherto been lacking.MethodsTo determine whether trientine treatment could improve in vivo outcome, we measured cardiac function in groups of trientine-treated diabetic (TETA-DIA), non-drug-treated diabetic (DIA) and sham-treated control (SHAM) rats, by using in vivo high-field cardiac magnetic-resonance imaging (cMRI) and an ex vivo isolated-perfused working heart method. Forty age-matched animals underwent a cMRI scan after which 12 were randomized to the SHAM group and 28 underwent streptozotocin-injection; of these, 25 developed stable diabetes, and 12 were then randomized to receive no treatment for 16 weeks (DIA) and the other 13 to undergo 8-weeks’ untreated diabetes followed by 8-weeks’ drug treatment (TETA-DIA). Animals were studied again by cMRI at 8 and 16 weeks following disease induction, and finally by measurement of ex vivo cardiac function.ResultsAfter eight weeks diabetes, rats (DIA/TETA-DIA) had developed significant impairment of LV function, as judged by impairment of ejection fraction (LVEF), cardiac output (CO), and LV mass (LVM)/body-mass (all P < 0.001), as well as other functional indexes. LVEF, CO (both P < 0.001) and the other indexes deteriorated further at 16 weeks in DIA, whereas trientine (TETA-DIA) improved cardiac function by elevating LVEF and CO (both P < 0.001), and also partially reversed the increase in LVM/body-mass (P < 0.05). In ex vivo hearts from DIA, the CO response to increasing preload pressure was deficient compared with SHAM (P < 0.001) whereas the preload-CO relationship was significantly improved in TETA-DIA animals (P < 0.001).ConclusionsTrientine treatment significantly improved cardiac function in diabetic rats with substantive LV impairment. These results implicate impaired copper regulation in the pathogenesis of impaired cardiac function caused by diabetic cardiomyopathy, and support ongoing studies of trientine treatment in patients with heart failure.

Aims/hypothesis Treatment with the Cu(II)-selective chelator triethylenetetramine (TETA) improves cardiovascular disease in human patients, and cardiac and vascular/renal disease in rats used as a model of diabetes. Here we tested two hypotheses: first, that TETA elicits greater improvement in organ function than less Cu-selective transition-metal-targeted treatments; second, that the therapeutic actions of TETA are consistent with mediation through suppression of oxidative stress.Methods Rats were made diabetic with streptozotocin (55 mg/kg, i. v.) and treated from 8 weeks after disease induction for the following 8 weeks with effective dosages of oral TETA, or one of three less Cu-selective transitionmetal-targeted treatments: D-penicillamine, deferiprone or Zn acetate. Treatment effects were measured in ex vivo cardiac and aortic tissues, plasma and urine. Results Diabetes damaged both cardiac and renal/vascular function by impairing the ability of cardiac output to respond physiologically to rising afterload, and by significantly elevatElectronic supplementary material The online version of this article

Renal depletion of myo-inositol (MI) is associated with the pathogenesis of diabetic nephropathy in animal models, but the underlying mechanisms involved are unclear. We hypothesized that MI depletion was due to changes in inositol metabolism and therefore examined the expression of genes regulating de novo biosynthesis, reabsorption, and catabolism of MI. We also extended the analyses from diabetes mellitus to animal models of dietary-induced obesity and hypertension. We found that renal MI depletion was pervasive across these three distinct disease states in the relative order: hypertension (-51%)>diabetes mellitus (-35%)>dietary-induced obesity (-19%). In 4-wk diabetic kidneys and in kidneys derived from insulin-resistant and hypertensive rats, MI depletion was correlated with activity of the MI-degrading enzyme myo-inositol oxygenase (MIOX). By contrast, there was decreased MIOX expression in 8-wk diabetic kidneys. Immunohistochemistry localized the MI-degrading pathway comprising MIOX and the glucuronate-xylulose (GX) pathway to the proximal tubules within the renal cortex. These findings indicate that MI depletion could reflect increased catabolism through MIOX and the GX pathway and implicate a common pathological mechanism contributing to renal oxidative stress in metabolic disease.

Hickey AJ, Chai CC, Choong SY, de Freitas Costa S, Skea GL, Phillips AR, Cooper GJ. Impaired ATP turnover and ADP supply depress cardiac mitochondrial respiration and elevate superoxide in nonfailing spontaneously hypertensive rat hearts. Am J Physiol Cell Physiol 297: C766 -C774, 2009. First published June 24, 2009 doi:10.1152/ajpcell.00111.2009.-Although most attention has been focused on mitochondrial ATP production and transfer in failing hearts, less has been focused on the nonfailing hypertensive heart. Here, energetic complications are less obvious, yet they may provide insight into disease ontogeny. We studied hearts from 12-mo-old spontaneously hypertensive rats (SHR) relative to normotensive Wistar-Kyoto (WKY) rats. The ex vivo working-heart model of SHR showed reduced compliance and impaired responses to increasing preloads. High-resolution respirometry showed higher state 3 (with excess ADP) respiration in SHR left ventricle fibers with complex I substrates and maximal uncoupled respiration with complex I ϩ complex II substrates. Respiration with ATP was depressed 15% in SHR fibers relative to WKY fibers, suggesting impaired ATP hydrolysis. This finding was consistent with a 50% depression of actomyosin ATPase activities. Superoxide production from SHR fibers was similar to that from WKY fibers respiring with ADP; however, it was increased by 15% with ATP. In addition, the apparent Km for ADP was 54% higher for SHR fibers, and assays conducted after ex vivo work showed a 28% depression of complex I in SHR, but not WKY, fibers. Transmission electron microscopy showed similar mitochondrial volumes but a decrease in the number of cristae in SHR mitochondria. Tissue lipid peroxidation was also 15% greater in SHR left ventricle. Overall, these data suggest that although cardiac mitochondria from nonfailing SHR hearts function marginally better than those from WKY hearts, they show dysfunction after intense work. Impaired ATP turnover in hard-working SHR hearts may starve cardiac mitochondria of ADP and elevate superoxide. adenylate control; saponin-permeabilized fibers; hypertensive heart; oxidative stress; complex I dysfunction; respiration MITOCHONDRIAL ENERGY PRODUCTION and linked energy transfer to ATP-consuming processes are central to the maintenance of physiological cell and tissue functions. Cardiac mitochondria occupy 30 -40% of the healthy mammalian cardiomyocyte (depending on species and health) and are regimentally arranged to supply 95% of cellular energy (for review see Ref. 20) via creatine kinase shuttle systems coupled to energy consumption sites (33,40,41). In hypertension-mediated hypertrophy and resulting heart failure, functional disruption occurs at all these levels (33), with changes in respiration (14,15,22,23,28,47), energy transfer (33, 53), and ATP consumption (55).In the context of heart failure (33), there has been a particular focus on mitochondria and energy transfer within the cardiomyocyte. This has driven many to study mitochondria in isolation and in vivo using NMR (4...

Heart disease is the major cause of death in diabetes, a disorder characterized by chronic hyperglycemia and cardiovascular complications. Diabetic cardiomyopathy (DCM) is increasingly recognized as a major contributor to diastolic dysfunction and heart failure in diabetes, but its molecular basis has remained obscure, in part because of its multifactorial origins. Here we employed comparative transcriptomic methods with quantitative verification of selected transcripts by reverse transcriptase quantitative PCR to characterize the molecular basis of DCM in rats with streptozotocin-induced diabetes of 16-wk duration. Diabetes caused left ventricular disease that was accompanied by significant changes in the expression of 1,614 genes, 749 of which had functions assignable by Gene Ontology classification. Genes corresponding to proteins expressed in mitochondria accounted for a disproportionate number of those whose expression was significantly modified in DCM, consistent with the idea that the mitochondrion is a key target of the pathogenic processes that cause myocardial disease in diabetes. Diabetes also induced global perturbations in the expression of genes regulating cardiac fatty acid metabolism, whose dysfunction is likely to play a key role in the promotion of oxidative stress, thereby contributing to the pathogenesis of diabetic myocardial disease. In particular, these data point to impaired regulation of mitochondrial beta-oxidation as central in the mechanisms that generate DCM pathogenesis. This study provides a comprehensive molecular snapshot of the processes leading to myocardial disease in diabetes.