Aysegul Durak

Hyperglycemia-induced changes in ZIP7 and ZnT7 expression cause Zn2+ release from the sarco(endo)plasmic reticulum and mediate ER-stress in the heart

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.

Interplay Between Cytosolic Free Zn(2+) and Mitochondrion Morphological Changes in Rat Ventricular Cardiomyocytes.

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-μM ZnPT minute applications by using confocal microscopy. We also showed that 1-μM ZnPT incubation induced significant increases in the phosphorylation levels of GSK3β (Ser21 and Ser9), Akt (Ser473), and NFκB (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.

Zn 2+ -transporters ZIP7 and ZnT7 play important role in progression of cardiac dysfunction via affecting sarco(endo)plasmic reticulum-mitochondria coupling in hyperglycemic cardiomyocytes

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, OPA1, 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.

Increased free Zn2+ correlates induction of sarco(endo)plasmic reticulum stress via altered expression levels of Zn2+-transporters in heart failure

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 Pearsons 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 PKCα expression and PKCα‐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 PKCα. 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 PKCα 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 PKCα can be important therapeutic approach in prevention/treatment of HF.

Cytosolic increased labile Zn2+ contributes to arrhythmogenic action potentials in left ventricular cardiomyocytes through protein thiol oxidation and cellular ATP depletion

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, Ito, steady-state currents, Iss and inward-rectifier K+-currents, IK1, 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 Iss and IK1, while no effect on Ito. However, the increased-[Zn2+]i could induce marked activation in ATP-sensitive K+-channel currents, IKATP, 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 IKATP 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 IKATP 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.

Aging related functional and structural changes in the heart and aorta: MitoTEMPO improves aged-cardiovascular performance

&NA; Aging in humans represents declining in cardio‐protective systems, however its mechanisms are not known yet. We aimed to analyse how aging affects key mechanisms responsible for contractile dysfunction via comparing the improperly synchrony between electrical and mechanical activities in male aged‐rats (24‐month old) comparison to those of adult‐rats (6‐month old). We determined significantly increased systemic oxidative stress with decreased antioxidant capacity, clear insulin resistance and hypertrophy in aged‐rats with normal fasting blood glucose. We also determined significantly high level of reactive oxygen species, ROS production in fluorescent dye chloromethyl‐2′,7′‐dichlorodihydrofluoroscein diacetate (DCFDA) loaded isolated cardiomyocytes from aged‐rats, confirming the increased oxidative stress in these hearts. In situ electrocardiograms, ECGs presented significant prolongations in RR‐ and QT‐intervals in the aged‐rats. Invasive hemodynamic measurements demonstrated marked increases in the heart rate and mean arterial pressure and decreases in the ejection‐fraction and preload‐recruitable stroke‐work, together with depressed contraction and relaxation activities in aortic rings. In light and electron microscopy examinations in aged‐rats, significant increases in muscle fibre radius and amount of collagen fibres were detected in the heart as well as markedly flattened and partial local splitting in elastic lamellas in the aorta, besides irregularly clustered mitochondria and lysosomes around the myofilaments in cardiomyocytes. MitoTEMPO treatment of tissue samples and cardiomyocytes from aged‐rats for 1‐h induced significant structural improvements. In the second part of our study, we have shown that mitochondria‐targeted antioxidant MitoTEMPO antagonized all alterations in the heart samples as well as penylephrine‐induced contractile and acetylcholine‐induced relaxation responses of aged‐rat aortic rings. Overall, the present data strongly support the important role of mitochondrial oxidative stress in the development of aged‐related insufficiencies and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in aging‐associated complications. Highlights Building upon our recent work in which, we examined whether a mitochondria‐targeted antioxidant MitoTEMPO could prevent cardiovascular dysfunction in an aged‐rat model and we showed for the first time that;We monitored insulin resistance and systemic oxidative status with decreased antioxidant capacity in aged‐rats.We observed a significant prolongation in RR‐ and QT‐intervals in ECG recordings.We determined a direct protection against cellular ROS production in isolated cardiomyocytes.We showed that improvement of mitochondrial antioxidant capacity with MitoTEMPO, Overall, the present data strongly support the important role of mitochondrial oxidative stress in the development of aged‐related insufficiencies and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in aging‐associated complications.

Onset of Depressed Heart Work is Correlated with the Increased Heart Rate and Shorten QT-Interval in High-Carbohydrate Fed Overweight Rats

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.