We examined whether cellular antioxidant-defence enhancement preserves diastolic dysfunction via regulation of both diastolic intracellular free Zn2+ and Ca2+ levels (
Diabetic cardiomyopathy was first recognized by Rubler et al. [
Accumulated evidence indicates that oxidative stress has closely been associated with diabetes and its complication including diabetic cardiomyopathy. Increased oxidative stress results also from a reduction of antioxidants/antioxidant defence system, thereby contributing to the initiation and progression of cardiac dysfunction [
Zinc, being an essential trace element, is vital in maintaining normal physiology and cellular functions in many cell types. Intracellularly, it is mostly bound to metalloproteins and plays a key role as an activating cofactor for many enzymes. On the cellular level, it has been demonstrated that the intracellular Zn2+ homeostasis is involved in signal transduction, in which Zn2+ acts as an intracellular mediator, similar to Ca2+ [
Zinc, a redox-inactive metal, has been long viewed as a component of the antioxidant network, and growing evidence points to its involvement in redox-regulated signaling via a direct or indirect regulation [
Male Wistar rats were used (200–250 g). Diabetes was induced in diabetic group as previously described [
Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and decompose to form a complex series of compounds, which include reactive carbonyl compounds, such as MDA. Plasma lipid peroxidation was determined by measuring malondialdehyde (MDA) level with thiobarbituric acid reactive substances (TBARS) assay kit (Cayman Chemical Company). The MDA-TBA adduct was formed under high temperature and acidic condition was measured colorimetrically at 530–540 nm.
Plasma glutathione status was determined by measuring reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, carried out with glutathione assay kit (Cayman Chemical Company) containing 2-(N-morpholino)ethanesulfonic acid buffer, GSSG standard, enzyme mixture, and 5,5′-dithiobis(2-nitrobenzoate). Levels of GSH and GSSG were calculated using reduced glutathione standard and the results were expressed as
To prepare heart homogenates, first frozen hearts were pulverized at liquid N2 temperature and then homogenized [
Total sulfhydryl (SH) and acid-soluble sulfhydryl (total thiol and free thiol) groups of proteins were estimated with Ellman’s reagent as described previously [
Histological examination was performed as described elsewhere. For electron microscopy evaluation, samples were fixed in 2.5% glutaraldehyde in phosphate buffer for 2–4 h at 4°C and postfixed in 1% osmium tetroxide. Following samples dehydration through graded alcohol concentrations (50%, 75%, 96%, and 100%), the heart tissues were embedded into Araldite 6005. The embedded samples were sliced at a thickness of 4–6
Hearts were isolated and perfused as described previously [
Cell isolation was performed as described elsewhere [
Fluorescence changes were recorded using microspectrophotometer and FELIX software (PTI, Lawrenceville, NJ, USA) as described previously [
Short-lived, tiny, and localized light emissions (named sparks) were recorded from different cardiomyocytes after 35–40 min incubation with FluoZin-3-AM or Fluo-3 AM, respectively, as described previously [
Sparks were initially detected with ImageJ (SparkMaster, plug-in) which is an open access programme (
For preparation of tissue homogenates, frozen heart samples from left ventricle were crushed at liquid N2 temperature and then homogenized to measure the phosphorylation and protein levels of contractile machinery complex as described previously [
Similar Western blot analysis was also performed in the diabetic rat cardiomyocytes from left ventricle incubated with
Unless otherwise stated, all chemicals used were purchased from Sigma (Sigma-Aldrich Chemie, Steinheim, Germany).
Groups were tested and compared using one-way ANOVA and Tukey post hoc test. Values of
A single administration of streptozotocin, STZ, to rats induced diabetic symptoms compared to the aged-matched controls including body weight loss (
Body weight, blood glucose, and oxidative stress markers of normal rats (CON group) and diabetic (DM) rats treated with
The extent of hyperglycemia-induced oxidative stress, lipid peroxidation level evaluated by MDA analysis in the heart homogenate, was significantly decreased in
Although diabetic rats did not show abnormal behaviour or any sign of heart failure as well as any signs of any tissue necrosis, as shown previously [
Morphological findings in experimental rat hearts. Micrographs of left ventricular cross section through control (CON), diabetic (DM), and
For quantification of electron microscopy findings among diabetic and nondiabetic rat hearts, we investigated only the lipid droplets and observed basically that the number and size of the lipid droplets in the diabetic group were significantly higher than that of the control group (~40% and ~15% for diabetic versus control group) while these changes have fully disappeared in the NAC-treated group. The lipid quantification of cardiac tissue was performed by using oil red O staining which revealed severe lipid accumulation in the heart. The lipid contents were expressed as lipid area normalized to total investigated tissue area.
We, previously, showed that STZ injection in rats either short- (4-5 weeks) or long-period (8–12 weeks) experimental durations induced marked several alterations in electrical and mechanical parameters of heart [
To identify and compare basal intracellular free Zn2+ level in diabetic rat cardiomyocytes with that of the control, we used a ratiometric fluorescence dye, Fura-2 AM. In order to compare the individual data, we normalized the initial fluorescence values to obtain a common ratio in all cardiomyocytes, and then we used TPEN responses for minimum and maximum fluorescence changes (
To test directly whether increased oxidative stress can induce simultaneous increases in basal levels of both intracellular free Zn2+ and Ca2+ of diabetic rat cardiomyocytes, we incubated cardiomyocytes from diabetic rats with NAC (1 mM, for 1 h at 37°C) before loading the cells with Fura-2 AM. As can be seen from Figure
Recently, we have demonstrated that there are local tiny Zn2+ releases (named as Zn2+ sparks) in resting quiescent cardiomyocytes isolated from 3-month-old male rats and loaded with a Zn2+-specific fluorescence dye, FluZin-3, which can be visualized in a similar manner to known Ca2+ sparks [
Of note, as shown in Figures
To further understand the effects of both diabetes and NAC treatment of diabetes on the distributions of both intracellular free Zn2+ and Ca2+ changes in isolated cardiomyocytes, we performed some additional experiments to monitor intracellular transient changes of either intracellular free Zn2+ or Ca2+ elicited by electrical-field stimulation. Figure
Altered intracellular global either Ca2+ or Zn2+ changes in cardiomyocytes are normalized with either
It has been previously shown that the mechanisms underlying the dysfunction of RyR2 in diabetes include, in part, a hyperphosphorylation of RyR2 due to both high phosphorylation levels of both protein kinase A (PKA) and Ca2+-calmodulin kinase II (CaMKII) under hyperglycemia [
Effect of
For an assessment of a direct action of antioxidant NAC, we first incubated freshly isolated cardiomyocytes from diabetic rats with 1 mM NAC for 1 h at 37°C and then performed the similar Western blot analysis for pRyR2, RyR2, FKBP12.6. Incubation of diabetic cardiomyocytes with 1 mM NAC significantly declined hyperphosphorylation level in RyR2, while there was no effect of its depressed protein level (Figure
The intracellular free Zn2+ distribution is linked to redox metabolism despite Zn2+ itself not being redox-active and generally Zn2+-proteins being redox-inert [
Direct effects of external Zn2+ on RyR2 macromolecular complex of ventricular heart tissue. (a)
There are multiple studies with different conclusions regarding whether NF-
This study on
The present study shows that systemic antioxidant treatment of diabetic rats preserves changes in both Zn2+ and Ca2+ regulation in diabetic cardiomyocytes without any effect on high blood glucose level and restores normal macromolecular complex composition and function of the RyR2 channels in diabetic rat hearts. There is associated restoration of myocardial diastolic function and reverse structural remodeling of the left ventricle in diabetic rats with
Taken together, these data suggest that one of the beneficial effects of NAC treatment, under
Notably, in here, we have shown that exogenously applied Zn2+ caused marked phosphorylation in RyR2 while there was no effect on the protein levels of both RyR2 and an accessory protein of RyR2 macromolecular complex, FKBP12.6, as well as higher phosphorphorylations in both PKA and CaMKII in a concentration-dependent manner, similar to hyperglycemia. These data are further supported, in part, with the fact that a Zn2+-binding protein calsequestrin resides in the SR and can bind up to 200 mol Zn2+ per mol, independently of binding up to 50 mol Ca2+ per mol calsequestrin. It is known as an association of Zn2+ with over 300 enzymes, where it can interact strongly with electronegative sulfur, nitrogen, and oxygen moieties in multiple coordination forms, serving catalytic and structural roles in maintaining active peptide conformations [
Therefore, it can be hypothesized that Zn2+ may compete with or substitute for metal ions crucial for the activity of signaling proteins. In addition to this hypothesis, it has been shown that Zn2+ has multiple functional effects on Ca2+/calmodulin-dependent protein kinase II (CaMKII) and modulates RyR1 binding to sarcoplasmic reticulum vesicles in skeletal muscle biphasically [
Thus, a major physiological role for Zn2+ may be the modulation of cell signaling cascades, especially those involving protein phosphorylation, and considering also that intracellular Zn2+ can rise quickly and can influence Ca2+ regulation [
Taken into consideration all present and also previously published data, in here, we report that an enhancement of antioxidant defence in diabetics, directly targeting heart, seems to prevent diastolic dysfunction, being associated with normalization of RyR2 macromolecular-complex, and thereby prevention of both Zn2+ and Ca2+ leaks leading to normalization of basal levels of intracellular free Ca2+ and Zn2+ in the heart. This is nicely in line with an early report performed on a rodent heart with hereditary muscular dystrophy, demonstrating accompany of intracellular Ca2+ overloading and oxidative stress with increased intracellular free Zn2+ [
No potential conflict of interests relevant to this paper was reported. Erkan Tuncay and Esma N. Okatan are equal co-first authors.
This work has been supported by grant from TUBITAK SBAG-111S042. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the paper. No additional external funding was received for this study. The authors would like to thank B. Can for her EM investigation.