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Zinc (mostly as free/labile Zn2+) is an essential structural constituent of many proteins, including enzymes in cellular signaling pathways via functioning as an important signaling molecule in mammalian cells. In cardiomyocytes at resting condition, intracellular labile Zn2+ concentration ([Zn2+]i) is in the nanomolar range, whereas it can increase dramatically under pathological conditions, including hyperglycemia, but the mechanisms that affect its subcellular redistribution is not clear. Therefore, overall, very little is known about the precise mechanisms controlling the intracellular distribution of labile Zn2+, particularly via Zn2+ transporters during cardiac function under both physiological and pathophysiological conditions. Literature data demonstrated that [Zn2+]i homeostasis in mammalian cells is primarily coordinated by Zn2+ transporters classified as ZnTs (SLC30A) and ZIPs (SLC39A). To identify the molecular mechanisms of diverse functions of labile Zn2+ in the heart, the recent studies focused on the discovery of subcellular localization of these Zn2+ transporters in parallel to the discovery of novel physiological functions of [Zn2+]i in cardiomyocytes. The present review summarizes the current understanding of the role of [Zn2+]i changes in cardiomyocytes under pathological conditions, and under high [Zn2+]i and how Zn2+ transporters are important for its subcellular redistribution. The emerging importance and the promise of some Zn2+ transporters for targeted cardiac therapy against pathological stimuli are also provided. Taken together, the review clearly outlines cellular control of cytosolic Zn2+ signaling by Zn2+ transporters, the role of Zn2+ transporters in heart function under hyperglycemia, the role of Zn2+ under increased oxidative stress and ER stress, and their roles in cancer are discussed.

Intracellular labile (free) Zn2+-level ([Zn2+](i)) is low and increases markedly under pathophysiological conditions in cardiomyocytes. High [Zn2+](i) is associated with alterations in excitability and ionic-conductances while exact mechanisms are not clarified yet. Therefore, we examined the elevated-[Zn2+](i) on some sarcolemmal ionic-mechanisms, which can mediate cardiomyocyte dysfunction. High-[Zn2+](i) induced significant changes in action potential (AP) parameters, including depolarization in resting membrane-potential and prolongations in AP-repolarizing phases. We detected also the time-dependent effects such as induction of spontaneous APs at the time of >= 3 min following [Zn2+](i) increases, a manner of cellular ATP dependent and reversible with disulfide-reducing agent dithiothreitol, DTT. High-[Zn2+](i) induced inhibitions in voltage-dependent K+-channel currents, such as transient outward K+-currents, I-to, steady-state currents, I-ss and inward-rectifier K+-currents, I-K1, reversible with DTT seemed to be responsible from the prolongations in APs. We, for the first time, demonstrated that lowering cellular ATP level induced significant decreaeses in both I-ss and I-K1, while no effect on I-to. However, the increased-[Zn2+](i) could induce marked activation in ATP-sensitive K+-channel currents, I-KATP, depending on low cellular ATP and thiol-oxidation levels of these channels. The mRNA levels of Kv4.3, Kv1.4 and Kv2.1 were depressed markedly with increased-[Zn2+](i) with no change in mRNA level of Kv4.2, while the mRNA level of I-KATP subunit, SUR2A was increased significantly with increased-[Zn2+](i), being reversible with DTT. Overall we demonstrated that high-[Zn2+](i), even if nanomolar levels, alters cardiac function via prolonged APs of cardiomyocytes, at most, due to inhibitions in voltage-dependent K+-currents, although activation of I-KATP is playing cardioprotective role, through some biochemical changes in cellular ATP- and thiol-oxidation levels. It seems, a well-controlled [Zn2+](i) can be novel therapeutic target for cardiac complications under pathological conditions including oxidative stress.

Objective Depressed mechanical activity is a marked complication in diabetics. Hypoxia has properties for novel diagnostic and therapeutic strategies, while intermittent hypoxia (IH) provides early functional and histologic remodeling, including some cardio benefits in early hemodynamic alterations with histologic remodeling and delayed changes in peripheral vasoreactivity. Therefore, we aimed to examine whether IH application presents a cardioprotective effect, via stabilization of hypoxia-inducible factor (HIF) in streptozotocin (STZ)-induced diabetic rat heart. Methods Male 10-week-old Wistar rats were randomly assigned as control group (C), IH group, (STZ)-induced diabetic group (DM) and IH applied DM group (DM+IH). Diabetes duration was kept 6 weeks and IH groups were exposed to hypobaric hypoxia at about 70 kPa (including ~14% PO2; 6 h/day for 6-weeks). Results Depressed left ventricular developed pressure (LVDP) and prolonged contraction and relaxation of Langendorff-perfused hearts, as well as increased total oxidative status from streptozotocin (STZ)-induced diabetic rats were markedly prevented with IH application. IH application induced significant increase in protein expression levels of both HIF-1α and vascular endothelial growth factor (VEGF), in both control and diabetic rat hearts, whereas there were significant decreases in the protein levels of prolyl-4 hydroxylase domain enzymes, PHD2, and PHD3 in diabetic hearts. Furthermore, IH application induced marked increases in protein levels of matrix metalloproteinases, MMP-2 and MMP-9 and capillary density in left ventricle of diabetic rats. Conclusion Overall, we presented how IH application has a beneficial cardiovascular remodeling effect in left ventricular function of diabetic rats, at most, via affecting increased oxidative stress and HIF-VEGF related angiogenesis, providing information on hyperglycemia associated new targets and therapeutic strategies.

Unfortunately, in the original publication of the article, Table 2 was published incorrectly. The correct version of Table 2 is provided in this correction.

Recent studies have implicated a relationship between RhoA/ROCK activity and defective Ca 2+ homeostasis in hypertrophic hearts. This study investigated molecular mechanism underlying ROCK inhibition-mediated cardioprotection against pressure overload-induced cardiac hypertrophy, with a focus on Ca 2+ homeostasis. Cardiac hypertrophy model was established by performing transverse aortic constriction (TAC) in 8-week-old male rats. Groups were assigned as SHAM, TAC and TAC + Fas (rats undergoing TAC and treated with fasudil). Rats in the TAC + Fas group were administered fasudil (5 mg/kg/day), and rats in the SHAM and TAC groups were treated with vehicle for 10 weeks. Electrophysiological recordings were obtained from isolated left ventricular myocytes and expression levels of proteins were determined using western blotting. Rats in the TAC group showed remarkable cardiac hypertrophy, and fasudil treatment significantly reversed this alteration. TAC + Fas myocytes showed significant improvement in reduced contractility and Ca 2+ transients. Moreover, these myocytes showed restoration of slow relaxation rate and Ca 2+ reuptake. Although L-type Ca 2+ currents did not change in TAC group, there was a significant reduction in the triggered Ca 2+ transients which was reversed either by long-term fasudil treatment or incubation of TAC myocytes with fasudil. The hearts of rats in the TAC group showed a significant decrease in ROCK1, ROCK2, RyR2 protein levels and p-PLB S16/T17 /SERCA2 ratio and increase in RhoA expression and MLC phosphorylation. However, fasudil treatment largely reversed TAC-induced alterations in protein expression. Thus, our findings indicate that upregulation of the RhoA/ROCK pathway is significantly associated with cardiac hypertrophy-related Ca 2+ dysregulation and suggest that ROCK inhibition prevents hypertrophic heart failure.

The cellular control of glucose uptake and glycogen metabolism in mammalian tissues is in part mediated through the regulation of protein-serine/threonine kinases including CK2. Although it participates to several cellular signaling processes, however, its subcellular localization is not well-defined while some documents mentioned its localization change under pathological conditions. The activation/phosphorylation of some proteins including Zn2+-transporter ZIP7 in cardiomyocytes is controlled with CK2α, thereby, inducing changes in the level of intracellular free Zn2+ ([Zn2+] i ). In this regard, we aimed to examine cellular localization of CK2α in cardiomyocytes and its possible subcellular migration under hyperglycemia. Our confocal imaging together with biochemical analysis in isolated sarco(endo)plasmic reticulum [S(E)R] and nuclear fractions from hearts have shown that CK2α localized highly to S(E)R and Golgi and weakly to nuclear fractions in physiological condition. However, it can migrate from nuclear fractions to S(E)R under hyperglycemia. This migration can further underlie phosphorylation of a target protein ZIP7 as well as some endogenous kinases and phosphatases including PKA, CaMKII, and PP2A. We also have shown that CK2α activation is responsible for hyperglycemia-associated [Zn2+] i increase in diabetic heart. Therefore, our present data demonstrated, for the first time, the physiological relevance of CK2α in cellular control of Zn2+-distribution via inducing ZIP7 phosphorylation and activation of these above endogenous actors in hyperglycemia/diabetes-associated cardiac dysfunction. Moreover, our present data also emphasized the multi-subcellular compartmental localizations of CK2α and a tightly regulation of these localizations in cardiomyocytes. Therefore, taken into consideration of all data, one can emphasize the important role of the subcellular localization of CK2α as a novel target-pathway for understanding of diabetic cardiomyopathy.

The death prevalence from cardiovascular disease is significantly high in elderly-populations, while mitochondrial-aging plays an important in abnormal function of vital organs through high mitochondrial ROS production. Mitochondria have a unique mode of action by providing ATP production and modulating the cytosolic Ca2+-signaling and maintain the redox status of cardiomyocytes. There is an aging-associated impairment in oxidative phosphorylation which causes a marked dysregulation of mitochondrial biogenesis. Therefore, we aimed to examine whether a mitochondria-targeting antioxidant, MitoTEMPO, can directly provide a cardioprotective effect on ventricular cardiomyocyte function under in vitro conditions. The MitoTEMPO-treatment (0.1 μM for 4-h) of aged-ventricular cardiomyocytes (from 24-mo-old rats), compared to those of the adults (from 8-mo-old rats) markedly augmented not only the depressed biochemical parameters but also the ultrastructure of mitochondria. It also provided marked protective action against increased mitochondrial superoxide formation and Bnip3 overexpression, which both markedly induce depolarized mitochondrial potential, increase reactive oxygen species, mitochondrial swelling and fission, and accelerate mitochondrial turnover via autophagy. Furthermore, it provided marked protection against spontaneous action potentials, via shortening the prolonged action potential duration, at most, through recovery in depressed K+-channel currents. Moreover, we determined significant recovery in the depressed intracellular Ca2+-changes under electrical stimulation in MitoTEMPO-treated the aged-cardiomyocytes. Overall, we provided important information associated with an antiarrhythmic action, thereby controlling cytosolic and mitochondrial Ca2+-handling, implying its possible protective role of mitochondria-targeting antioxidant-treatment during aging.

Ticagrelor, a P2Y12-receptor inhibitor, and a non-thienopyridine agent are used to treat diabetic patients via its effects on off-target mechanisms. However, the exact sub-cellular mechanisms by which ticagrelor exerts those effects remains to be elucidated. Accordingly, the present study aimed to examine whether ticagrelor influences directly the cardiomyocytes function under insulin resistance through affecting mitochondria-sarco(endo)plasmic reticulum (SER) cross-talk. Therefore, we analyzed the function and ultrastructure of mitochondria and SER in insulin resistance-mimicked (50-mu M palmitic acid for 24-h) H9c2 cardiomyocytes in the presence or absence of ticagrelor (1-mu M for 24-h). We found that ticagrelor treatment significantly prevented depolarization of mitochondrial membrane potential and increases in reactive oxygen species with a marked increase in the ATP level in insulin-resistant H9c2 cells. Ticagrelor treatment also reversed the increases in the resting level of free Ca2+ and mRNA level of P2Y12 receptors as well as preserved ER stress and apoptosis in insulin-resistant H9c2 cells. Furthermore, we determined marked repression with ticagrelor treatment in the increased number of autophagosomes and degeneration of mitochondrion, including swelling and loss of crista besides recoveries in enlargement and irregularity seen in SER in insulin-resistant H9c2 cells. Moreover, ticagrelor treatment could prevent the altered mRNA levels of Becklin-1 and type 1 equilibrative nucleoside transporter (ENT1), which are parallel to the preservation of ultrastructural ones. Our overall data demonstrated that ticagrelor can directly affect cardiomyocytes and provide marked protection against ER stress and dramatic induction of autophagosomes, and therefore, can alleviate the ER stress-induced oxidative stress increase and cell apoptosis during insulin resistance.

Redox signalling in mitochondria plays an important role in myocardial ischaemia/reperfusion (I/R) injury and in cardioprotection. Reactive oxygen and nitrogen species (ROS/RNS) modify cellular structures and functions by means of covalent changes in proteins including among others S‐nitros(yl)ation by nitric oxide (NO) and its derivatives, and S‐sulphydration by hydrogen sulphide (H2S). Many enzymes are involved in the mitochondrial formation and handling of ROS, NO and H2S under physiological and pathological conditions. In particular, the balance between formation and removal of reactive species is impaired during I/R favouring their accumulation. Therefore, various interventions aimed at decreasing mitochondrial ROS accumulation have been developed and have shown cardioprotective effects in experimental settings. However, ROS, NO and H2S play also a role in endogenous cardioprotection, as in the case of ischaemic pre‐conditioning, so that preventing their increase might hamper self‐defence mechanisms. The aim of the present review was to provide a critical analysis of formation and role of reactive species, NO and H2S in mitochondria, with a special emphasis on mechanisms of injury and protection that determine the fate of hearts subjected to I/R. The elucidation of the signalling pathways of ROS, NO and H2S is likely to reveal novel molecular targets for cardioprotection that could be modulated by pharmacological agents to prevent I/R injury.

Redox signalling in mitochondria plays an important role in myocardial ischaemia/reperfusion (I/R) injury and in cardioprotection. Reactive oxygen and nitrogen species (ROS/RNS) modify cellular structures and functions by means of covalent changes in proteins including among others S‐nitros(yl)ation by nitric oxide (NO) and its derivatives, and S‐sulphydration by hydrogen sulphide (H2S). Many enzymes are involved in the mitochondrial formation and handling of ROS, NO and H2S under physiological and pathological conditions. In particular, the balance between formation and removal of reactive species is impaired during I/R favouring their accumulation. Therefore, various interventions aimed at decreasing mitochondrial ROS accumulation have been developed and have shown cardioprotective effects in experimental settings. However, ROS, NO and H2S play also a role in endogenous cardioprotection, as in the case of ischaemic pre‐conditioning, so that preventing their increase might hamper self‐defence mechanisms. The aim of the present review was to provide a critical analysis of formation and role of reactive species, NO and H2S in mitochondria, with a special emphasis on mechanisms of injury and protection that determine the fate of hearts subjected to I/R. The elucidation of the signalling pathways of ROS, NO and H2S is likely to reveal novel molecular targets for cardioprotection that could be modulated by pharmacological agents to prevent I/R injury.

Metabolic syndrome (MetS) is increasingly common among humans all over the world and a combination of serious pathological conditions occurred together. MetS induces increasing of risks for several organ dysfunctions including heart and type 2 diabetes (T2DM) since it is closely linked to overweight or obesity and inactivity among humans, currently, also linking to insulin resistance. To prevent MetS, it is needed first to have a healthy lifestyle, however, several therapeutic approaches also are in used to lighten its risky effects. Sodium-glucose co-transporter 2 (SGLT2) and 1 (SGLT1) inhibitors are relatively new glucose-lowering agents that work by increasing urinary glucose excretion through the kidneys, exerting their action independently of insulin. However, there are a number of side effects of these agents in humans with MetS. Nevertheless, different research teams, recently, demonstrated that SGLT2 inhibitors (SGLT2is) exert important cardioprotective effects in patients with MetS and T2DM via lowering the high risks for cardiovascular morbidity and mortality. Furthermore, it has been also emphasized that SGLT2is-associated cardioprotection in insulin-resistant overweights rats includes prevention of prolongation in ventricular-repolarization via marked augmentation of mitochondrial function together with normalization of oxidative stress followed by improvement of fusion-fission proteins, without its glucose-lowering effect. Moreover, two recent clinical studies announced that SGLT2is, electrophysiologically, could provide marked protective effects on electrocardiographic parameters in T2DM patients. Therefore, in the present review article, it has been documented the recent data related to SGLT2is on both experimental and clinical studies and their outcomes in terms of either adverse, beneficial, or both effects.

The prevalence of death from cardiovascular disease is significantly higher in elderly populations; the underlying factors that contribute to the age‐associated decline in cardiac performance are poorly understood. Herein, we identify the involvement of sodium/glucose co‐transporter gene (SGLT2) in disrupted cellular Ca2+‐homeostasis, and mitochondrial dysfunction in age‐associated cardiac dysfunction. In contrast to younger rats (6‐month of age), older rats (24‐month of age) exhibited severe cardiac ultrastructural defects, including deformed, fragmented mitochondria with high electron densities. Cardiomyocytes isolated from aged rats demonstrated increased reactive oxygen species (ROS), loss of mitochondrial membrane potential and altered mitochondrial dynamics, compared with younger controls. Moreover, mitochondrial defects were accompanied by mitochondrial and cytosolic Ca2+ ([Ca2+]i) overload, indicative of disrupted cellular Ca2+‐homeostasis. Interestingly, increased [Ca2+]i coincided with decreased phosphorylation of phospholamban (PLB) and contractility. Aged‐cardiomyocytes also displayed high Na+/Ca2+‐exchanger (NCX) activity and blood glucose levels compared with young‐controls. Interestingly, the protein level of SGLT2 was dramatically increased in the aged cardiomyocytes. Moreover, SGLT2 inhibition was sufficient to restore age‐associated defects in [Ca2+]i‐homeostasis, PLB phosphorylation, NCX activity and mitochondrial Ca2+‐loading. Hence, the present data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility through a mechanism that impinges upon [Ca2+]i‐homeostasis. Our studies support the notion that interventions that modulate SGLT2‐activity can provide benefits in maintaining [Ca2+]i and cardiac function with advanced age.

Insulin resistance, impaired glucose regulation, dyslipidemia, low-grade inflammation, and elevated blood pressure are main components of the metabolic syndrome (MetS). Trace elements, especially zinc (Zn) and copper (Cu) and cytokines, have physiological importance due to their presence in inflammatory processes and glucose metabolism. Therefore, this study aimed to investigate the potential relationship between cytokine responses and trace elements in different tissues of sucrose-induced MetS rats compared with healthy controls (n:7/groups). Tissue Zn concentrations are found to be decreased in the liver (p = 0.00) and pancreas (p

Obesity-induced insulin resistance and type 2 diabetes mellitus can ultimately result in various complications, including diabetic cardiomyopathy. In this case, cardiac dysfunction is characterized by metabolic disturbances such as impaired glucose oxidation and an increased reliance on fatty acid oxidation. Mitochondrial dysfunction has often been associated with the altered metabolic function in the diabetic heart, and may result from fatty acid-induced lipotoxicity and uncoupling of oxidative phosphorylation. In this review, we address the metabolic changes in the diabetic heart, focusing on the loss of metabolic flexibility and cardiac mitochondrial function. We consider the alterations observed in mitochondrial substrate utilization, bioenergetics and dynamics, and highlight new areas of research which may improve our understanding of the cause and effect of cardiac mitochondrial dysfunction in diabetes. Finally, we explore how lifestyle (nutrition and exercise) and pharmacological interventions can prevent and treat metabolic and mitochondrial dysfunction in diabetes.

Excitation-contraction coupling in normal cardiac function is performed with well balanced and coordinated functioning but with complex dynamic interactions between functionally connected membrane ionic currents. However, their genomic investigations provide essential information on the regulation of diseases by their transcripts. Therefore, we examined the gene expression levels of the most important voltage-gated ionic channels such as Na

Myocardial contractility is controlled by intracellular Ca2+ cycling with the contribution of sarcoplasmic reticulum (SR). In this study, we aimed to investigate the role of altered SR function in defective regulation of intracellular Ca2+ levels in rats with metabolic syndrome (MetS) induced by a 16-week high-sucrose drinking-water diet. Electric-field stimulated transient intracellular Ca2+ changes in MetS cardiomyocytes exhibited significantly reduced amplitude (∼30%) and prolonged time courses (2-fold), as well as depressed SR Ca2+ loading (∼55%) with increased basal Ca2+ level. Consistent with these data, altered ryanodine receptor (RyR2) function and SERCA2a activity were found in MetS cardiomyocytes through Ca2+ spark measurements and caffeine application assay in a state in which sodium calcium exchanger was inhibited. Furthermore, tetracaine application assay results and hyperphosphorylated level of RyR2 also support the “leaky RyR2” hypothesis. Moreover, altered phosphorylation levels of phospholamban (PLN) support the depressed SERCA2a-activity thesis and these alterations in the phosphorylation of Ca2+-handling proteins are correlated with altered protein kinase and phosphatase activity in MetS cardiomyocytes. In conclusion, MetS-rat heart exhibits altered Ca2+ signaling largely due to altered SR function via changes in RyR2 and SERCA2a activity. These results point to RyR2 and SERCA2a as potential pharmacological targets for restoring intracellular Ca2+ homeostasis and, thereby, combatting dysfunction in MetS-rat heart.

The Zn2+ in cardiomyocytes is buffered by structures near T-tubulus and/or sarcoplasmic/endoplasmic reticulum (S(E)R) while playing roles as either an antioxidant or a toxic agent, depending on the concentration. Therefore, we aimed first to examine a direct effect of ZnPO4 (extracellular exposure) or Zn2+ pyrithione (ZnPT) (intracellular exposure) application on the structure of the mitochondrion in ventricular cardiomyocytes by using histological investigations. The light microscopy data demonstrated that Zn2+ exposure induced marked increases on cellular surface area, an indication of hypertrophy, in a concentration-dependent manner. Furthermore, a whole-cell patch-clamp measurement of cell capacitance also supported the hypertrophy in the cells. We observed marked increases in mitochondrial matrix/cristae area and matrix volume together with increased lysosome numbers in ZnPO4- or ZnPT-incubated cells by using transmission electron microscopy, again in a concentration-dependent manner. Furthermore, we observed notable clustering and vacuolated mitochondrion, markedly disrupted and damaged myofibrils, and electron-dense small granules in Zn2+-exposed cells together with some implications of fission-fusion defects in the mitochondria. Moreover, we observed marked depolarization in mitochondrial membrane potential during 1-mu M ZnPT minute applications by using confocal microscopy. We also showed that 1-mu M ZnPT incubation induced significant increases in the phosphorylation levels of GSK3 beta (Ser21 and Ser9), Akt (Ser473), and NF kappa B (Ser276 and Thr254) together with increased expression levels in ER stress proteins such as GRP78 and calregulin. Furthermore, a new key player at ER-mitochondria sites, promyelocytic leukemia protein (PML) level, was markedly increased in ZnPT-incubated cells. As a summary, our present data suggest that increased cytosolic free Zn2+ can induce marked alterations in mitochondrion morphology as well as depolarization in mitochondrion membrane potential and changes in some cytosolic signaling proteins as well as a defect in ER-mitochondria cross talk.

Aging is an important risk factor for cardiac dysfunction. Heart during aging exhibits a depressed mechanical activity, at least, through mitochondria-originated increases in ROS. Previously, we also have shown a close relationship between increased ROS and cellular intracellular free Zn2+ ([Zn2+]i) in cardiomyocytes under pathological conditions as well as the contribution of some re-expressed levels of Zn2+-transporters for redistribution of [Zn2+]i among suborganelles. Therefore, we first examined the cellular (total) [Zn2+] and then determined the protein expression levels of Zn2+-transporters in freshly isolated ventricular cardiomyocytes from 24-month rat heart compared to those of 6-month rats. The [Zn2+]i in the aged-cardiomyocytes was increased, at most, due to increased ZIP7 and ZnT8 with decreased levels of ZIP8 and ZnT7. To examine redistribution of the cellular [Zn2+]i among suborganelles, such as Sarco/endoplasmic reticulum, S(E)R, and mitochondria ([Zn2+]SER and [Zn2+]Mit), a cell model (with galactose) to mimic the aged-cell in rat ventricular cell line H9c2 was used and demonstrated that there were significant increases in [Zn2+]Mit with decreases in [Zn2+]SER. In addition, the re-distribution of these Zn2+-transporters were markedly changed in mitochondria (increases in ZnT7 and ZnT8 with no changes in ZIP7 and ZIP8) and S(E)R (increase in ZIP7 and decrease in ZnT7 with no changes in both ZIP8 and ZnT8) both of them isolated from freshly isolated ventricular cardiomyocytes from aged-rats. Furthermore, we demonstrated that cellular levels of ROS, both total and mitochondrial lysine acetylation (K-Acetylation), and protein-thiol oxidation were significantly high in aged-cardiomyocytes from 24-month old rats. Using a mitochondrial-targeting antioxidant, MitoTEMPO (1 &micro, M, 5-h incubation), we provided an important data associated with the role of mitochondrial-ROS production in the [Zn2+]i-dyshomeostasis of the ventricular cardiomyocytes from 24-month old rats. Overall, our present data, for the first time, demonstrated that a direct mitochondria-targeting antioxidant treatment can be a new therapeutic strategy during aging in the heart through a well-controlled [Zn2+] distribution among cytosol and suborganelles with altered expression levels of the Zn2+-transporters.

Zinc is an important micronutrient for mammalians, whereas free/labile zinc ion (Zn2+) level ([Zn2+]i) in cells can be detrimental if it is over the physiological range. The cellular [Zn2+]i is regulated by proteins, responsible for its influx (ZIP family) into cells or efflux (ZnT-family) from cells. In addition, it has been shown that there is a close relationship between cellular [Zn2+]i and increasing oxidative stress, and both are also responsible for the dysregulation of the excitation/contraction coupling in the heart. Although age-dependent changes in the structure and function of the left ventricle are closely related to fibrosis, cellular evidence showed the importance of mitochondria as a potential target for aging medicine. However, several studies have shown that zinc supplementation provides important improvements in the maintenance of cardiovascular disorders, and it is needed to know the exact role of [Zn2+]i and its transporters in aging heart function. Although there is very little information related to the role of Zn2+ transporters in mammalian aging, some studies have shown their importance in cardiovascular disorders including failing human heart. Since few studies examined the role of ZIP14 as an inflammation responsive transporter and amplified with aging, we demonstrated an important data on the ZIP14 status in organelles such as the sarcoplasmic reticulum and mitochondria in aging rat’s ventricular cardiomyocytes. As a summary, we discussed our data in the light of literature data as well.

Three major biological roles of zinc (as Zn2+) in several proteins and enzymes, including the catalytic, structural and regulatory, brings Zn2+ to be an important signaling molecule. Zn2+ mediates many vital physiological processes in mammalian cells, including normal cellular growth, synthesis of DNA, and stability of membranes. Since Zn2+ has hydrophilic property, its direct passive diffusion does not possible through cell membranes. Transportation of Zn2+ in both directions through the cell membrane, therefore, needs special transport systems such as specific Zn2+-transporters, besides others binding or sensing Zn2+. Although recent studies focused on the biological role of free (labile) Zn2+ ([Zn2+]i), its functional role in cardiomyocytes, at the cellular level, is not completely understood. Having a strong relation with cellular [Ca2+]i, the [Zn2+]i in cardiomyocytes found to be increased markedly under both hyperglycemia and oxidative stress. Recently, several important data pointed out the vital role of cellular well-controlled [Zn2+]i through Zn2+-transporters in cardiomyocytes Zn2+ signaling by Zn2+-transporters in normal heart function. Furthermore, those studies emphasized how any alteration in Zn2+ signaling due to changes in either [Zn2+]i and/or function of Zn2+-transporters can underline the development of pathological conditions in the heart. Taken into consideration the importance of Zn2+-transporters in the regulation of [Zn2+]i in cardiomyocytes, the present review focused on documentation of the present and recent data on the hypothesis of possible relations between high [Zn2+]i and oxidative stress in heart dysfunction under hyperglycemia.

Functional contribution of S(E)R-mitochondria coupling to normal cellular processes is crucial and any alteration in S(E)R-mitochondria axis may be responsible for the onset of diseases. Mitochondrial free Zn2+ level in cardiomyocytes ([Zn2+](Mit)) is lower comparison to either its cytosolic or S(E)R level under physiological condition. However, there is little information about distribution of Zn2+-transporters on mitochondria and role of Zn2+-dependent mitochondrial-function associated with [Zn2+](Mit). Since we recently have shown how hyperglycemia (HG)-induced changes in ZIP7 and ZnT7 contribute to Zn2+-transport across S(E)R and contribute to S(E)R-stress in the heart, herein, we hypothesized that these transporters can also be localized to mitochondria and affect the S(E)R-mitochondria coupling, and thereby contribute to cellular Zn2+-muffling between S(E)R-mitochondria in HG-cells. Mitochondrial localizations of ZIP7 and ZnT7 were demonstrated using fluorescence technique while they were confirmed in isolated mitochondrial fractions using biochemical analysis. Markedly decreased ZIP7 and increased ZnT7 levels were measured in isolated mitochondrial fractions from either HG- or doxorubicin, DOX (as positive control)-treated cardiomyocytes. Significantly increases in [Zn2+](Mit) and ROS production levels and depolarized mitochondrial membrane potential were also measured in HG cells. The expression levels of some key proteins, responsible for proper S(E)R-mitochondria coupling such as Mfn-1, Fis-1, OPAl, BAP31, STIM1 and PML in either HG- or DOX-cells were supported our above hypothesis, strongly. Overall, this study provides an important description about the role of ZIP7 and ZnT7, localized to both mitochondria and S(E)R and contribute to cellular Zn2+-muffling between cellular-compartments in HG or hypertrophic cardiomyocytes via affecting S(E)R-mitochondria coupling. Any alteration in this axis and/or cellular [Zn2+) may provide new insight for prevention/therapy of HF in diabetes and/or hypertrophy.

Background: the study aimed to investigate whether timolol-treatment has a beneficial effect on pentose phosphate pathway enzyme activities such as glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGDH) enzyme activities and cAMP level in streptozotocin-induced diabetic rats in pancreatic tissues. Methods: diabetes was induced by streptozotocin (STZ) in 3-month old male Wistar rats. The diabetic rats were treated with timolol (5 mg/kg body weight, for 12 weeks) while the control group received saline. Enzyme activities were determined in pancreas tissue. To support our results, we performed in silico calculations, using Protein Data Bank structures. Results: timolol treatment of STZ-induced diabetic rats had no noteworthy effect on high blood-glucose levels. However, this treatment induced activities of G6PD and 6PGDH in diabetic rats. Timolol treatment significantly increased cAMP level in diabetic pancreatic tissue. We found that timolol cannot bind strongly to either G6PD or 6PGD, but there is a relatively higher binding affinity to adenylyl cyclase, responsible for cAMP production, serving as a regulatory signal via specific cAMP-binding proteins. Conclusions: our data point out that timolol treatment has beneficial effects on the antioxidant defence mechanism enzymes in the pancreas of STZ-induced diabetic rats. / Uvod: cilj istrazivanja je bio da se utvrdi da li tretman timololom ima pozitivan efekat na aktivnosti enzima pentoze fosfata, kao sto su aktivnosti glukoze-6-fosfat dehidrogenaze (G6PD), enzimske aktivnosti 6-fosfoglukonat dehidrogenaze i cAMP nivo u tkivu pankreasa kod pacova kojima je dijabetes izazvan streptozotocionom. Metode: dijabetes je izazvan streptozotocionom (STZ) kod tromesecnih muzjaka vistar pacova. Pacovi sa dijabetesom su tretirani timololom (5 mg/kg telesne tezine tokom 12 nedelja), dok je kontrolna grupa primila fizioloski rastvor. Enzimske aktivnosti su utvrivane u tkivu pankreasa. Da bismo potkrepili nase rezultate, sproveli smo in silico racunanja koristeci strukture Proteinske baze podataka. Rezultati: tretman timololom na pacovima kojima je dijabetes izazvan putem STZ-a nije imao znacajan uticaj na visoke nivoe glukoze u krvi. Medutim, kod takvih pacova ovaj tretman je indukovao aktivnosti G6PD i 6PGDH. Lecenje timololom znacajno je povecalo nivo cAMP-a u dijabeticnom tkivu pankreasa. Utvrdili smo da se timolol ne moze snazno vezati ni za G6PD, ni za 6PGD, ali da postoji relativno veci afinitet vezivanja za adenilil ciklazu, odgovornu za proizvodnju cAMP, koja sluzi kao regulatorni signal putem odredenih cAMP vezivnih proteina. Zakljucak: nasi podaci ukazuju da tretman timololom ima pozitivne efekte na antioksidantne enzime od brambenog sistema u pankreasu pacova sa dijebetesom izazvanim putem STZ-a., Research Infrastructure Support Program of the Ministry of Development of the Republic of Turkey

Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed significant effects in patients with diabetes or metabolic syndrome (MetS) with high cardiovascular risk. Although the increased intracellular Zn

Role of β

Metabolic syndrome, is associated impaired blood glucose level, insulin resistance, and dyslipidemia caused by abdominal obesity. Also, it is related with cardiovascular risk accumulation and cardiomyopathy. The hypothesis of this study was to examine the effect of thiazolidinediones such as pioglitazone on intracellular Na

PubMedID: 26521868 The aim of this study was to investigate the association between metabolic syndrome (MetS) and the metabolic indicators of masticatory muscles in an animal model. A total of 16 male Wistar rats were used. To induce MetS, 10 rats were fed with standard rat chow and 32% sucrose solution ad libitum for 16 wk. Six rats fed with standard rat chow and water ad libitum formed the control group. All rats were killed at week 16, and the right superficial masseter muscles were harvested. Metabolic indicators of masticatory muscle metabolism, including antioxidant enzyme activities, ion transport ATPase activities, and the levels of macro and trace elements, were determined in the muscles. Superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities were significantly decreased by 32%, 26%, 33%, and 16%, respectively, in the MetS group. Na+/K+-ATPase activity was significantly decreased in the MetS group by 54% compared with the control group. The levels of chromium and selenium were significantly decreased, and the level of copper was increased, in the MetS group compared with the control group. These results show that significant alterations occurred in antioxidant defense mechanisms, ion transport mechanisms, and trace element levels of masseter muscles in MetS. © 2015 European Journal of Oral Sciences.

Trace elements can participate in the catalysis of group-transfer reactions and can serve as their structural components. However, most of them including zinc and selenium have multifunctional roles in biological environments such as antioxidant and/or pro-oxidant effects, as concentration-dependent manner. Although it has been demonstrated the antioxidant actions of either selenium or zinc compounds, there are several documents pointing out their pro-oxidant/oxidant roles in biological systems. Here we have used ELISA-based immuno-spin trapping, a method for detection of free radical formation, to detect whether or not a zinc compound, Zn3(PO4)2, or a selenium compound, Na2SeO3, has antioxidant and/or pro-oxidant effect on 5,5-Dimethyl-1-Pyrroline-N-Oxide (DMPO)-DNA nitrone adducts induced with Cu(II)-H2O2-oxidizing system in in vitro preparations. Second, we examined whether this technique is capable to demonstrate the different DMPO-protein nitrone adduct productions in isolated protein crude of hearts from normal rats (CON) or rats with metabolic syndrome (MetS). Our data demonstrated that either Zn(2+) (100 µM) or SeO3(-2) (50 nM) has very strong antioxidant action against 200 µM H2O2-induced DMPO-DNA nitrone adduct production, whereas their higher concentrations have apparent pro-oxidant actions. We also used verification by Western blotting analysis whether immuno-spin trapping can be used to assess H2O2-induced DMPO-protein nitrone adducts in heart protein crudes. Our Western blot data further confirmed the ELISA-data from proteins and demonstrated how Zn(2+) or SeO3(-2) are dual-functioning ions such as antioxidant at lower concentrations while pro-oxidant at higher concentrations. Particularly, our present data with SeO3(-2) in DMPO-protein nitrone adducts, being in line with our previous observation on its dual-actions in ischemia/reperfusion-induced damaged heart, have shown that this ion has higher pro-oxidant actions over 50 nM in MetS-group compared to that of CON group.

Little is known about metabolic syndrome (MetS)-associated cardiomyopathy, especially in relation to the role and contribution of beta-adrenoceptor (β-AR) subtypes. Therefore, we examined the roles of β-AR subtypes in the cardiac function of rats with MetS (MetS group) and compared it with that of rats with streptozotocin (STZ)-induced diabetes (STZ group). Compared with the normal control rats, the protein levels of cardiac β1- and β2-AR in the MetS group were significantly decreased and with no changes in their mRNA levels, whereas the protein levels of β3-AR were similar to those of the controls. However, as shown previously, the protein levels of cardiac β1- and β2-AR in the STZ group were decreased, whereas the β3-AR levels were significantly increased by comparison with the controls. Additionally, the mRNA levels of β2- and β3-AR were increased, but β1-AR mRNA was decreased in the STZ group. Furthermore, left ventricular developed pressure responses to β3-AR agonist BRL37344 were increased in the STZ group but not in the MetS group, whereas for both groups, the responses to noradrenaline were not different from those of the controls. However, the response to stimulation with high concentrations of fenoterol was depressed in the MetS group, compared with the controls, but not in the STZ group. Consequently, our data suggest that the contribution of the β-AR system to cardiac dysfunction in the rats with MetS is not the same as that in the STZ group, although they have similar cardiac dysfunction with similar ultrastructural changes to the myocardium.

Objective: Metabolic syndrome (MetS), generally characterized with insulin-resistance, leads to severe cardiovascular disorders, however, the underlying mechanisms of this syndrome has not yet been fully determined. In addition, there are some controversies related with unti-diabetic agents used for treat-ment of MetS-induced complications, including their aggravating effects on the depressed cardiac function. Therefore, in this study, we first aimed to investigate the effects of pioglitazone, known as a insulin-sensitizer, on MetS-induced heart dysfunction. Second, for a comparative study to determine whether MetS-induced heart dysfunction is due to increased oxidative stress, we used a known antioxidant (querce-tin) to MetS rats for the same period.Material and Methods: Total 28 male Wistar rats (3 months) were grouped as control group, (Con), MetS group (MetS; added 32% sucrose to drinking water for 18-week), MetS group supplemented with pioglita-zone (Piog; 30 mg/kg/day via gavage for 2-week, from 16th-week of MetS-group) and MetS with quercetin (Quer; 75 mg/kg/day via gavage for 2-weeks from 16th-week of MetS group). All rats were fed with standard rat-diet and tap water at standard condition. At the end of experimental period, the body weights and blood glucose levels, and total oxidative status (TOS) and total antioxidant status (TAS) in serum by using special kits were measured for all animals. By using Langendorff-isolated-organ system, left ventricle pressu-re changes (LVPC) were measured in isolated hearts. The basal level of intracellular free Ca2+ ([Ca2+]i) and Ca2+ transients under electrical-situmulation (under action-potential) were measured in freshly isolated cardiomyocytes by enzymatic-method (loaded with Ca2+-sensitive fluorescence dye, Fura-2AM).Results and Discussion: Either pioglitazone or quercetin application have positive effects on body weight and high blood sugar levels in MetS-group rats. Additionally, both agents caused a small but significant increase on serum TAS levels (p

Zn2+-homoeostasis including free Zn2+ ({[}Zn2+](i)) is regulated through Zn2+-transporters and their comprehensive understanding may be important due to their contributions to cardiac dysfunction. Herein, we aimed to examine a possible role of Zn2+-transporters in the development of heart failure (HF) via induction of ER stress. We first showed localizations of ZIP8, ZIP14 and ZnT8 to both sarcolemma and S(E)R in ventricular cardiomyocytes (H9c2 cells) using confocal together with calculated Pearson's coefficients. The expressions of ZIP14 and ZnT8 were significantly increased with decreased ZIP8 level in HF. Moreover, {[}Zn2+](i) was significantly high in doxorubicin-treated H9c2 cells compared to their controls. We found elevated levels of ER stress markers, GRP78 and CHOP/Gadd153, confirming the existence of ER stress. Furthermore, we measured markedly increased total PKC and PKC alpha expression and PKC alpha-phosphorylation in HF. A PKC inhibition induced significant decrease in expressions of these ER stress markers compared to controls. Interestingly, direct increase in {[}Zn2+](i) using zinc-ionophore induced significant increase in these markers. On the other hand, when we induced ER stress directly with tunicamycin, we could not observe any effect on expression levels of these Zn2+ transporters. Additionally, increased {[}Zn2+](i) could induce marked activation of PKC alpha. Moreover, we observed marked decrease in {[}Zn2+](i) under PKC inhibition in H9c2 cells. Overall, our present data suggest possible role of Zn2+ transporters on an intersection pathway with increased {[}Zn2+](i) and PKC alpha activation and induction of HF, most probably via development of ER stress. Therefore, our present data provide novel information how a well-controlled {[}Zn2+](i) via Zn2+ transporters and PKC alpha can be important therapeutic approach in prevention/treatment of HF.

Clinical and experimental studies have shown an association between intracellular free Zn2+ ([Zn2+](i))-dyshomeostasis and cardiac dysfunction besides [Ca2+](i)-dyshomeostasis. Since [Zn2+](i)-homeostasis is regulated through Zn2+-transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn2+-homeostasis via alteration in Zn2+-transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3 beta, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NF kappa B to the NF kappa B are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKC alpha and PKC alpha levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn2+-influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn2+](i) detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn2+](i) via decreased ER-[Zn2+] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn2+-transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn2+](i)- besides [Ca2+](i)-dyshomeostasis.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

Functional contribution of S(E)R-mitochondria coupling to normal cellular processes is crucial and any alteration in S(E)R-mitochondria axis may be responsible for the onset of diseases. Mitochondrial free Zn

Mechanical activity of the heart is adversely affected in metabolic syndrome (MetS) characterized by increased body mass and marked insulin resistance. Herein, we examined the effects of high carbohydrate intake on cardiac function abnormalities by evaluating in situ heart work, heart rate, and electrocardiograms (ECGs) in rats. MetS was induced in male Wistar rats by adding 32% sucrose to drinking water for 22–24 weeks and was confirmed by insulin resistance, increased body weight, increased blood glucose and serum insulin, and increased systolic and diastolic blood pressures in addition to significant loss of left ventricular integrity and increased connective tissue around myofibrils. Analysis of in situ ECG recordings showed a markedly shortened QT interval and decreased QRS amplitude with increased heart rate. We also observed increased oxidative stress and decreased antioxidant defense characterized by decreases in serum total thiol level and attenuated paraoxonase and arylesterase activities. Our data indicate that increased heart rate and a shortened QT interval concomitant with higher left ventricular developed pressure in response to β-adrenoreceptor stimulation as a result of less cyclic AMP release could be regarded as a natural compensation mechanism in overweight rats with MetS. In addition to the persistent insulin resistance and obesity associated with MetS, one should consider the decreased heart work, increased heart rate, and shortened QT interval associated with high carbohydrate intake, which may have more deleterious effects on the mammalian heart.

Clinical and experimental studies have shown an association between intracellular free Zn

Recent studies have implicated a relationship between RhoA/ROCK activity and defective Ca

Changes in cellular free Zn2+ concentration, including those in the sarco(endo)plasmic reticulum [S(E)R], are primarily coordinated by Zn2+ transporters (ZnTs) whose identity and role in the heart are not well established. We hypothesized that ZIP7 and ZnT7 transport Zn2+ in opposing directions across the S(E)R membrane in cardiomyocytes and that changes in their activity play an important role in the development of ER stress during hyperglycemia. The subcellular S(E)R localization of ZIP7 and ZnT7 was determined in cardiomyocytes and in isolated S(E)R preparations. Markedly increased mRNA and protein levels of ZIP7 were observed in ventricular cardiomyocytes from diabetic rats or high-glucose–treated H9c2 cells while ZnT7 expression was low. In addition, we observed increased ZIP7 phosphorylation in response to high glucose in vivo and in vitro. By using recombinant-targeted Förster resonance energy transfer sensors, we show that hyperglycemia induces a marked redistribution of cellular free Zn2+, increasing cytosolic free Zn2+ and lowering free Zn2+ in the S(E)R. These changes involve alterations in ZIP7 phosphorylation and were suppressed by small interfering RNA–mediated silencing of CK2α. Opposing changes in the expression of ZIP7 and ZnT7 were also observed in hyperglycemia. We conclude that subcellular free Zn2+ redistribution in the hyperglycemic heart, resulting from altered ZIP7 and ZnT7 activity, contributes to cardiac dysfunction in diabetes.

Zinc (Zn2+) ions are increasingly recognized as playing an important role in cellular physiology. Whereas the free Zn2+ concentration in the cytosol has been established to be 0.1-1 nM, the free Zn2+ concentration in subcellular organelles is not well-established. Here, we extend the eCALWY family of genetically encoded Forster Resonance Energy Transfer (FRET) Zn2+ probes to permit measurements in the endo(sarco)plasmic reticulum (ER) and mitochondria! matrix. Deployed in a variety of mammalian cell types, these probes reveal resting mitochondrial free [Zn2+] values of similar to 300 pM, somewhat lower than in the cytosol but 3 orders of magnitude higher than recently reported using an alternative FRET-based sensor. By contrast, free ER [Zn2+] was found to be >= 5 nM, which is >= 5000-fold higher than recently reported but consistent with the proposed role of the ER as a mobilizable Zn2+ store. Treatment of beta-cells or cardiomyocytes with sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors, mobilization of ER Ca2+ after purinergic stimulation with ATP, or manipulation of ER redox, exerted no detectable effects on [Zn2+](ER). These findings question the previously proposed role of Ca2+ in Zn2+ mobilization from the ER and suggest that high ER Zn2+ levels may be an important aspect of cellular homeostasis.

High glucose and increased oxidative stress levels are the known important mediators of diabetic nephropathy. However, the effects of these mediators on tissue damage basically due to extracellular matrix expansion in mesangial cells have yet to be fully examined within the context of early stage diabetic nephropathy. In this study, we attempted to characterize changes in mesangial cells of streptozotocin-induced diabetic rats with a comparative investigation of kidney tissue by using different microscopy techniques. The serum levels of urea and creatinine of diabetic rats, as biomarkers of kidney degeneration, decreased significantly compared to those of age-matched controls. In diabetic rats, there are increased malondialdehyde and oxidized-glutathione levels as well as reduced-glutathione and glutathione-peroxidase activity levels in renal tissue compared to those of the controls. By using light and electron microscopies, we showed that there were marked thickening in Bowman's membrane and glomerular capillary wall, increased amount of extracellular matrix often occupying Bowman's space, degenerations in tubules, an increased number of mesangial cells in the network of glomerular capillary walls, and increased amount of lipid accumulation in proximal tubules in the renal tissue of diabetic rats. Our confocal microscopy data confirmed also the presence of irregularity and widened in glomerular capillaries, their attachment to the Bowman's capsule, degenerated heterochromatin, thickening in foci of glomerular basement membrane, and marked increase in mesangial cells. These results suggest that a detailed structural investigation of kidney tissue provides further information on the important role of mesangial cells in pathogenesis of diabetic nephropathy.

Changes in cellular free Zn

The scope of this review is to summarize the important roles of vitamin E family members as protective agents in cardiovascular pathologies of different types of disease states and particularly in diabetes, including some of our research results, to illustrate how this recent knowledge is helping to better understand the roles of the vitamin E family in biology, in animals and humans specifically. Cardiovascular disease, a general name for a wide variety of diseases, disorders and conditions, is caused by disorders of the heart and blood vessels. Cardiovascular disease is the world's largest killer, claiming 17.1 million lives a year. Cardiovascular complications result from multiple parameters including glucotoxicity, lipotoxicity, fibrosis. Obesity and diabetes mellitus are also often linked to cardiovascular disease. In fact, cardiovascular disease is the most life-threatening of the diabetic complications and diabetics are 2- to 4-fold more likely to die of cardiovascular-related causes than non-diabetics. In order to prevent the tendency of cardiovascular disease, primary prevention is needed by modifying risk factors. Several recent studies, besides earlier ones, have reported beneficial effects of therapy with antioxidant agents, including trace elements, vitamins (E and/or C), other antioxidants, against the cardiovascular dysfunction. Hence, the use of peroxisome proliferator activated receptor-α (PPARα) agonists to reduce fatty acid oxidation, of trace elements such as selenium as antioxidant and other antioxidants such as vitamins E and C, contributes to the prevention of these dysfunctions. Moreover, therapy with antioxidants and the above vitamins to prevent or delay the onset and development of cardiovascular complications in diabetic patients and animal models has been investigated although these studies showed inconsistent results.

Increasing evidence shows a marked beneficial effect with β-blockers in heart dysfunction via scavenging reactive oxygen species. Previously we showed that chronic treatment with either timolol or propranolol possessed similar beneficial effects for heart function in male rats as age increased, whereas only timolol exerted similar benefits in female rats. Therefore, in this study, we aimed first to examine the cellular bases for age-related alterations in excitation–contraction coupling in ventricular myocytes from female rats and, second, to investigate the hypothesis that age-related changes in [Ca2+]ihomeostasis and receptor-mediated system can be prevented with chronic timolol treatment. Chronic timolol treatment of 3-month-old female rats abolished age-related decrease in left ventricular developed pressure and the attenuated responses to β-adrenoreceptor stimulation. It also normalized the altered parameters of [Ca2+]itransients, decreased Ca2+loading of sarcoplasmic reticulum and increased basal [Ca2+]i, and decreased L-type Ca2+currents in 12-month-old female rats compared with the 3-month-old group. Adenylyl cyclase activity, β-adrenoreceptor affinity to its agonist, and β-adrenoreceptor density of the 12-month-old group are normalized to those of the 3-month-old group. Moreover, timolol treatment prevented dysfunction of the antioxidant system, including increased lipid peroxidation, decreased ratio of reduced glutathione to oxidized glutathione, and decreased activities of thioredoxin reductase and glucose-6-phosphate dehydrogenase, in the left ventricle of hearts from the 12-month-old group. Our data confirmed that aging-related early myocardial impairment is primarily related to a dysfunctional antioxidant system and impairment of Ca2+homeostasis, which can be prevented with chronic timolol treatment.

Studies have shown that tetracycline class antibiotics exhibit an ameliorating action with its antioxidant property on increased oxidative stress in tissues, including heart. Since endothelial vascular dysfunction in diabetes is associated with increased oxidative stress and prevented with antioxidants, herein, we aimed to test a hypothesis whether a low-dose doxycycline treatment of diabetic rats for 4 weeks can ameliorate endothelial vascular dysfunction of thoracic aortas. Results of the present study shows that both direct and alpha receptor-mediated contractile responses as well as endothelium-dependent and endothelium-independent vasodilatory responses were preserved with low-dose doxycycline treatment (30 μmol/kg, daily; for 4 weeks) compared with untreated diabetic group. Furthermore, doxycycline treatment normalized increased lipid peroxidation and cellular glutathione level measured in plasma and prevented diabetes-induced impaired body weight gain without significant effect on high blood glucose level. Increased membrane protein level of caveolin-1, elevated ratio of PKC in particulate and cytosolic fraction, and increased protein level of cytosolic endothelin-1 in diabetic rats were also significantly prevented with doxycycline treatment. Moreover, diabetes-induced another type of oxidative stress markers in rats, matrix metalloproteinases, MMP-2, and MMP-9 were also normalized with doxycycline treatment in blood. Taken together, our data address that amelioration and/or prevention of vascular endothelial and contractile dysfunction by doxycycline is accompanied by a clear reduction in oxidative stress markers of diabetes, which provides evidence for doxycycline's potential antioxidant action as a therapeutic agent for amelioration and/or prevention of vascular disorders in diabetic subjects.

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