Here we report that a marked deficiency in total copper, of ~50%, occurred in the LV myocardium of diabetic rats with DCM and, strikingly, that there was full restoration of copper to control levels following treatment with the Cu (II)-selective chelator, TETA, [8, 48]. We also demonstrate that TETA-mediated restoration of LV copper was accompanied by marked improvement in the structural and functional defects in the LV of rats with DCM, consistent with prior reports [8, 48, 60]. TETA treatment with the dosage used for the current protocol did not have any adverse effects on cardiac function in non-diabetic control LV, although long-term treatment with higher dosages could be expected to cause symptomatic copper deficiency . We also identified several myocellular copper proteins that respond to TETA treatment in diabetes, but TETA treatment in non-diabetic controls did not alter expression of copper transporters CTR1 and CTR2. Therefore TETA treatment at the dosage employed does not affect copper transport, and further data from TETA-treated non-diabetic controls are not required for interpretation of results of the current study. One question that then arises from these data is that of how copper-chelation successfully ameliorates LV copper deficiency in diabetes?
Physiological copper exists in the body in two valence states, Cu (I) which is mainly localized in the intracellular compartment and comprises ~95% of total body-copper, and Cu (II), which is largely present in the extracellular space and comprises the remaining ~5% . We report here that diabetic rats have lower myocardial copper, a result mainly of decreased intracellular Cu (I), whereas we have shown previously that chelatable extracellular cardiac Cu (II) is increased by ~3-fold in diabetic rats . These results support the hypothesis that the copper-deficient state in the diabetic LV-myocardium is associated with insufficient [Cu (I)]IC but excessive [Cu (II)]EC: that is, it reflects an imbalanced distribution of myocardial copper consistent with defective copper uptake into the LV. Heart failure in diabetes may thus be explained, at least in part, by disruption of the distribution of the two copper valence states, and the concomitant myocardial damage that ensues.
Elevated [Cu (II)]EC is thought to promote the increased formation of advanced glycated end-products (AGEs) in collagen, and enhanced TGF-β-evoked collagen deposition . TETA is a selective Cu (II) chelator [45, 64] that binds free copper, thus suppressing Cu (II)-catalyzed reactions of reactive oxygen species, such as O2●- or H2O2, to generate HO● radicals in the ECM . Therefore, the improvement in cardiac structure and function caused by TETA treatment may ultimately be evoked, at least in part, by the rebalancing of the intracellular-to-extracellular ratio of the copper valence states. However, little is known of the molecular basis for regulation of relative myocardial Cu (I)/Cu (II) distribution. The molecular mechanisms through which TETA may correct this imbalance were thus a major focus of this study.
Normal cardiac structure and function is reportedly sensitive to marginal copper deficiency: as copper deficiency worsens, the heart is said to become more susceptible to oxidative damage through impairment of its copper-dependent defense mechanisms, such as those catalyzed by SOD1 . Here we have investigated the mechanisms through which impaired cellular copper regulation could alter the biology of proteins in the copper pathways and their potential influence on the LV myocardium. We have provided compelling evidence for dysregulation of cellular copper pathways in the LV in DCM. Key alterations include: (i) decreased expression of the high-affinity copper transporter CTR1; (ii) decreased expression and increased polymerization of the copper-binding/anti-oxidant defense proteins MT1/2; (iii) decreased levels of CCS and SOD1, coupled with diminished SOD1 activity; (iv) decreased expression of ATOX1 and ATP7B, which could well impair copper transport to sites of synthesis of copper-enzymes within the secretory pathway; and (v) translocation of ATP7B, which may alter copper efflux from or between cardiomyocytes.
The diminished expression of myocardial CTR1 protein levels provides a potential mechanism for localized copper deficiency in the diabetic LV. Unaltered Ctr1 mRNA levels are consistent with the idea that this copper deficiency may well be generated at a translational rather than transcriptional level. The findings of deficient total copper (mainly Cu (I)) in the LV coupled with substantively diminished CTR1 at the cell membrane, and with increased internalisation and lowered overall levels of the transporter, are consistent with a mechanism known to occur in several mammalian cell types, namely copper-evoked endocytosis [65, 66] and degradation : these findings are consistent with the presence of elevated Cu (II) in the ECM of the coronary vasculature in the diabetic heart, which could well trigger this defect  and thus cause heart failure in diabetes . The disorganised pattern of CTR1 distribution in the T-tubular region of the diabetic LV is also noteworthy. Consistently, it has been reported that ventricular myocytes from diabetic animals with heart failure possess a sparse, irregular T-tubule system : it is possible that this disorganization could contribute to impaired myocardial copper uptake. Furthermore, the T-tubules are an important determinant of cardiomyocyte function, especially as they are the main site of excitation-contraction coupling . Therefore the observed structural changes at this subcellular site of CTR1 localization may point to a link between defective myocardial copper uptake and impaired myocardial contractility [9, 10], implying a potential role of myocellular copper homeostasis in the regulation of excitation-contraction coupling: however, myocellular copper currently has no known role in myocardial excitation-contraction coupling . By contrast, since TETA treatment did not correct diminished CTR1 levels in diabetes, CTR1 is unlikely to play a role in the TETA-evoked correction of LV copper levels and function.
Contrastingly, TETA treatment increased the expression of Ctr2 mRNA and protein compared to untreated diabetic values but not to untreated control values, suggesting the effects of TETA on these pathways occurred only in diabetes. TETA treatment also enhanced CTR2 localization at the cell periphery, specifically at the outer sarcolemmal membrane and the intercalated disk region, where it could increase copper uptake from the extracellular space and neighboring cardiomyocytes, respectively. Therefore, elevation of CTR2 is a candidate mechanism whereby TETA can restore copper levels in diabetic LV. There is evidence that cells possess more than one copper uptake pathway. Thus, dietary copper supplementation of pregnant mice did not rescue Ctr1−/− offspring, suggesting that Ctr1−/− embryos cannot acquire copper because of the lack of the plasma membrane CTR1 transporter; however, CTR1-deficient mouse embryonic cells possess a second, CTR1-independent copper transport system . CTR2 localizes in part to the cell membrane, and cells lacking CTR2 have lower copper accumulation . Therefore, although CTR2 is a lower-affinity copper transporter than CTR1, TETA may nevertheless correct LV-copper levels by up-regulating CTR2, thereby increasing copper import. Moreover, TETA-evoked increase in the sarcolemmal localization of CTR2 contrasts with the observed enhancement of CTR2 localization in the vesicular compartments in diabetic LV: a vesicular localization for CTR2 has previously been reported, where it was noted to co-localize with both lysosomes and late endosomes . Diabetes-mediated elevations in CTR2 expression in vesicular membranes are possibly the endogenous compensatory response by which copper released from copper proteins by lysosomal degradation is recycled into the cytosol and thus made available for cellular utilization in response to lowered copper uptake by CTR1: this could happen without changing total cellular copper levels. Moreover, the elevated recycling of copper into the cytosol via CTR2 could also serve as a signal of increasing intracellular copper levels, in turn further lowering copper uptake by CTR1. Thus CTR2 and CTR1 show opposing changes in expression and thus, quite possibly, opposing roles in diabetes-induced LV-copper deficiency. Lastly, our findings in diabetic rats contrast with the lowered expression of CTR2 in the hearts of rats with diet-induced systemic copper-deficiency : this contrast points to a distinct pathogenic mechanism in the regulation of copper uptake in the LV myocardium of the diabetic rat in the context of the overall systemic copper overload that occurs in diabetes [8, 45].
Metallothioneins are one of the major classes of copper-binding proteins contributing to the regulation of intracellular copper homeostasis and protection against excess cytoplasmic copper [61, 72], acting through their actions as a scavenger of transition metal atoms and radicals . Previous studies have implied that oxidative stress induced by chronic hyperglycemia can impair intracellular copper homeostasis in the diabetic heart, in part by suppressing myocardial MT expression [61, 73]. Here, we detected decreased expression but increased polymerization of MT in diabetic myocardium which may lead to decreased availability of redox-responsive forms of MT, and thus to decreased protection against copper-mediated toxicity. The lowering of total MT will lead to a concomitant reduction in cytoplasmic copper-binding capacity, which is consistent with the lowered intracellular copper levels we observed, most likely attributable to reduced CTR1-mediated copper influx. The decreased expression of the highly polymerized, ~70-kD isoform of MT present after TETA treatment is consistent with the decrease in levels of copper-containing MT associated with decreased cellular oxidation. This finding correlates with improved functional activities of MT, and is consistent with a process of MT-mediated correction in myocardial cytoplasmic copper levels.
Here we also found evidence of decreased copper supply to SOD1 via CCS in diabetic myocardium, which could lead to the measured deficiencies in SOD1 activity. The catalytic function of SOD1 is dependent on copper redox chemistry at its active site, and is thus potentially regulated by rates of cellular copper supply: the observed relative inactivity of SOD1 in diabetic myocardium is consistent with deficient copper metalation of apo-SOD1. It has been reported that SOD1 with lowered copper content is less catalytically active, and may also be unstable and degraded faster than the normally-metalated enzyme . The decreased activity of SOD1 could lead to diminished anti-oxidant protection thus enabling enhanced oxidative damage in the diabetic LV-myocardium. In contrast to other reports showing up-regulation of CCS protein caused by dietary copper-deficiency in rats , our study demonstrates lowered levels of CCS in copper-deficient LV myocardium, consistent with a contrasting role of CCS in response to the changes of copper status under diabetic conditions. TETA treatment rectified CCS levels and the activity of SOD1, consistent with restoration of copper-supply to SOD1 via CCS, reversing deficient SOD1 activity, and contributing to demonstrated restoration of myocardial anti-oxidant defenses .
ATP7A and ATP7B contribute to the maintenance of copper-dependent enzyme activity by delivering copper to the lumen of the secretory pathway in the trans-Golgi network, where copper metalation generates active cuproenzymes. These ATPases can also contribute to the maintenance of intracellular copper concentrations by transporting copper into secretory vesicles from the trans-Golgi network, which shuttle to the plasma membrane for copper excretion . Here we found that, in diabetic LV, protein levels of both ATOX1 and ATP7B were decreased, consistent with possible impairment of copper supply for activation of cuproenzymes. The decreased localization of ATP7B to the sarcolemmal membrane and intercalated disc regions in diabetes could limit intercellular transport of copper. Cumulatively these mechanisms could contribute to the pattern of copper deficiency and impaired cardiac function [9, 10]. We also found that the ATP7A copper transporter, which is thought to function mainly in intestinal copper acquisition , is expressed in the myocardium. TETA enhanced the expression of ATP7A mRNA and protein, which could compensate and help to rectify impaired copper delivery to the secretory pathway/intercellular transport by defective ATP7B action. These effects could contribute to the restoration of physiological copper homeostasis and biosynthesis of active copper-dependent enzymes in the myocardium, in parallel to the TETA-mediated restoration of the CCS-SOD1 pathway. The observed up-regulation of ATP7A with enhanced peri-nuclear localization following TETA treatment is consistent with enhanced delivery of copper to the trans-Golgi network, presumably aiding copper supply for newly-synthesized copper proteins.
Some of the observed abnormalities, for example lowered levels of CTR1, CCS and SOD1, probably contribute to the causation of the localized copper-deficiency state in diabetic myocardium or to its adverse consequences, and thus to the functional impairment observed in the hearts of diabetic animals [8, 45, 48]; other effects, such as elevated CTR2 and lowered MT1/2 and ATP7B, may reflect endogenous responses directed towards ameliorating copper deficiency or its impacts.
How might TETA exert its intracellular effects? We have previously shown that TETA selectively binds excess Cu (II) in diabetic individuals and elicits its removal from the body via urinary excretion, through studies where the following methods were applied: electron paramagnetic resonance spectroscopy; X-ray crystallography; potentiometric, spectrophotometric and mass-spectrometric analysis of complex formation between Cu (II), and TETA and its metabolites; and clinical studies [8, 45, 55, 64]. Thus TETA can remove excess Cu (II) from the ECM, probably by binding and removing it from pathogenic binding sites such as those in AGE-modified collagen [77, 78]. AGE-coordinated Cu (II) almost certainly remains catalytically active, and could therefore bind to the external, high-affinity Cu (II)-binding site present near the NH2-terminus of CTR1 . Thus, elevated Cu (II) bound to AGE-modified collagen in diabetic individuals could participate in the modulation of cell copper metabolism through binding to CTR1, perhaps resulting in its translocation away from the cell membrane as shown herein. However, TETA is also known to cross cell membranes, probably via mechanisms employed by its physiological homologues, spermine and spermidine . For example, there is substantive evidence that TETA can traverse cell membranes in the gut and kidney via a Na+/spermine-antiporter-mediated mechanism . It thus has the potential to exert direct effects in the intracellular compartment.
TETA forms two main metabolites in the body, monoacetyl-TETA and diacetyl-TETA [54, 82–85]. Both acetyl metabolites are strong chelators in their own right, although their affinities for Cu (II) are substantially less than that of the parent compound . There is evidence that monoacetyl-TETA may contribute to the overall response to treatment in diabetic patients . There are no published reports known to us, however, describing the uptake of TETA directly into cardiomyocytes, so whether TETA and its metabolites could act directly within the intracellular compartment to influence copper homeostasis remains to be determined.
These studies have also provided a new molecular explanation linking the therapeutic effects of TETA in diabetes to the restoration of myocardial copper content. We provide new evidence of intracellular targets of TETA treatment, whose amended expression/activity restore antioxidant defenses, which include: (i) decreasing the polymerization of MT1/2, consistent with diminished pro-oxidant stress; (ii) restoring LV-myocardial copper uptake, at least in part via increased expression of CTR2 in the sarcolemma; (iii) increasing copper supply to SOD1 via increased expression of CCS, leading to restoration of SOD1 function; (iv) increasing expression and localization of ATP7A in the trans-Golgi region, by which it could improve copper translocation into the lumen of the secretory pathway for synthesis of active cuproenzymes. Our results indicate that hyperglycemia-induced cell-copper imbalance in cardiomyocytes might be rectified by TETA treatment via increased sarcolemmal copper importation coupled with compensatory modifications in the export pathway. Cu (II)-chelation with TETA treatment could thus allow the (partial) restoration of copper delivery to intracellular sites of utilization and storage.