Mitochondria-Targeting Antioxidant Provides Cardioprotection Through

International Journal of Molecular Sciences

Article Mitochondria-Targeting Antioxidant Provides Cardioprotection through Regulation of Cytosolic and Mitochondrial Zn2+ Levels with Re-Distribution of Zn2+-Transporters in Aged Rat Cardiomyocytes

Yusuf Olgar, Erkan Tuncay and Belma Turan * Departments of Biophysics, Ankara University Faculty of Medicine, 06100 Ankara, Turkey * Correspondence: [email protected]; Tel.: +90-312-5958186; Fax: +90-312-3106370

Received: 21 May 2019; Accepted: 17 June 2019; Published: 2 August 2019

Abstract: 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 2+ 2+ Zn ([Zn ]i) in cardiomyocytes under pathological conditions as well as the contribution of some 2+ 2+ re-expressed levels of Zn -transporters for redistribution of [Zn ]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 2+ to those of 6-month rats. The [Zn ]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 2+ cellular [Zn ]i among suborganelles, such as Sarco/endoplasmic reticulum, S(E)R, and mitochondria 2+ 2+ ([Zn ]SER and [Zn ]Mit), a cell model (with galactose) to mimic the aged-cell in rat ventricular 2+ cell line H9c2 was used and demonstrated that there were significant increases in [Zn ]Mit with 2+ 2+ decreases in [Zn ]SER. In addition, the re-distribution of these Zn -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 µM, 5-h incubation), we provided an important 2+ data associated with the role of mitochondrial-ROS production in the [Zn ]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.

Keywords: aging-heart; intracellular free zinc; zinc-transporters; cardiovascular function; oxidative stress; insulin resistance; mitochondria

1. Introduction Aging in humans is programmed genetically and modified by environmental influences as well as having a combination of morphological and functional changes in organs, tissues, and cells [1]. The rate of aged human percentage among populations is accelerating all over the world. The effect of aging can vary widely from one to another individual [2]. High resistance to blood pumping and increased workload against the increased resistance are the general findings in aged individuals [3].

Int. J. Mol. Sci. 2019, 20, 3783; doi:10.3390/ijms20153783 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 3783 2 of 18

With a short statement, human aging is a time-related relapse of the physiological functions during the life span, including insufficient cardiovascular functioning. Recent and even early both clinical and experimental studies mentioned that QT-intervals in electrocardiogram were prolonged with age in an overtly healthy older population [4–7]. Furthermore, there are a number of studies demonstrating a relationship between insulin resistance and cardiac dysfunction in elderly individuals and experimental animals [6,8–10]. Indeed, one component of aging-associated development of cardiac dysfunction is alteration in cardiac energy metabolism [11–13]. Moreover, an important amount of literature data documented that there is a close link, which shows the involvement of oxidative stress in the pathogenesis of aging. The production of an increasing amount of lipid peroxidation and its accumulation into the heart during aging can increase the susceptibility to the development of cardiovascular diseases besides others, at most, due to the production of reactive oxygen species (ROS) and free radicals [6,14,15]. More importantly, there are early studies demonstrating the role of zinc in biological samples, associated with dietary zinc and the levels of lipid peroxidation as well as protein thiol oxidation in the mammalian tissues [16,17]. At the cellular level, there are several damaging changes in cardiomyocytes such as marked increases in their dimensions, decreases in the number of living and functional cardiomyocytes [6]. Furthermore, in terms of cellular electrical activities, among other observations, there were significant action potential prolongation, changes in ionic fluxes across sarcolemma, and dysregulation in 2+ 2+ intracellular free Ca level ([Ca ]i), mitochondrial defects and increased oxidative stress in cardiomyocytes [11,18–22]. Although there is not yet a consensus on the role of mitochondria in aging-associated insufficient cardiomyocyte function, mitochondria-associated increases in intracellular ROS seems to play a central role during physiological mammalian aging. It is well accepted that the main role of mitochondria is to provide ATP demand and modulate the cytosolic Ca2+-signaling, influencing cellular ROS, and regulating the redox state of cells [23–25]. On the other hand, it has been also demonstrated that exposing oxidants could cause a very important increase in cytosolic free Zn2+ 2+ 2+ level in cardiomyocytes ([Zn ]i), comparable to that of [Ca ]i [26]. Furthermore, we have recently 2+ + shown that the high [Zn ]i could induce marked activation in ATP-sensitive K -channel currents, depending on the cellular ATP levels [27], while a depletion in ATP production in insulin-resistant rat ventricular cardiomyocytes could also induce significant prolongation in action potentials [6,28]. All of these already known to imply a cross-correlation between insulin resistance, alterations in 2+ [Zn ]i and cardiac dysfunction in the aged rat cardiomyocytes. Moreover, there is also a strong link between mitochondrial metabolism, ROS generation, and the senescent state. In this regard, early studies demonstrated also that mitochondrial mutations increase in frequency with age in both animal models and in humans [29,30]. Furthermore, since a depolarization-induced Zn2+-uptake induced 2+ 2+ the high amount of cell death and enhanced accumulation of mitochondrial free Ca ([Ca ]Mit) in Ca2+- and Zn2+-containing media, the interactive roles of Zn2+ and Ca2+ in mitochondrial dysfunction have been studied and demonstrated in neural studies [31]. Moreover, these studies also showed 2+ how increased [Ca ]Mit led to important impairments in different intracellular signal transduction pathways, including cellular oxidative stress and apoptosis [2,32,33]. In general, zinc, being a multipurpose element for the mammalian body, plays important roles for the regulation of several cellular signaling mechanisms as Zn2+ [34]. Although there are limited numbers 2+ of data existing in the field of the role of [Zn ]i in the aged heart, few studies have demonstrated 2+ 2+ the role of alterations in [Zn ]i-homeostasis, at least, via alterations in Zn -transporting proteins (ZIP and ZnT families). In this regard, it has been widely discussed the importance of mitochondrial Ca2+-homeostasis as potential target event for mitochondrial medicine [35,36]. 2+ 2+ The [Zn ]i-homeostasis and the suborganelle levels of [Zn ] are altered under pathological conditions, including hyperglycemia in cardiomyocytes [37,38]. Moreover, we have shown that a re-distribution of the expression level of some Zn2+-transporters can play an important contribution to those changes [37,39]. In the literature, there is a limited number of studies on the role of cellular [Zn2+] and Zn2+-transporters in the aged heart function [40]. In this field, authors demonstrated the Int. J. Mol. Sci. 2019, 20, 3783 3 of 18 fact of ZIP14 role as an inflammation responsive transporter and it has important phenotypic effects, amplified with aging [41]. Therefore, in the present study, we first aimed to examine the distribution of 2+ 2+ 2+ cellular [Zn ] among suborganelles, such as S(E)R and mitochondria ([Zn ]SER and [Zn ]Mit) in ventricular cardiomyocytes from aged rats compared to young ones (24-month vs. 6-month). Second, we examined a possible contribution of redistribution of Zn2+-transporters among suborganelles in these aged cardiomyocytes. Third, using a mitochondrial-targeting antioxidant MitoTEMPO, we examined the important contribution of mitochondria to the aging-associated changes into aged cardiomyocyte dysfunction.

2. Results

2.1. General Parameters of the Aged Rats The average body weights of the aged and young rats were 380 12 g and 310 8 g, respectively. ± ± The heart to body weight ratio was higher in the 24-month old rats (aged group; number of rats: 10) than the 6-month old rats (young group; number of rats: 9), significantly (5.5 0.3 mg/g vs. 4.2 0.1 mg/g), ± ± indicating the slightly but significantly development of hypertrophy. The fasting blood glucose level was also slightly but significantly high in the old rats compared to the young rats (93 3 mg/dL vs. ± 82 2 mg/dL). As shown previously, there was a significant presence of insulin resistance in the old ± group with the measurement of oral glucose tolerance test (OGTT) and homeostasis model assesment of of insulin resistance (HOMA-IR) index, as described previously [6]. Although the aged group has a similar fasting blood glucose level with a young group, they have about a 17% high blood glucose level during 30-min of OGTT and that increased level during 60, 90, and 120 min measurements was still high, by about 10% compared to those of the young group. The HOMA-IR index was measured as 7.8 1.3 in the aged groups and 6.1 1.0 in the young group, in which the differences between these two ± ± groups were significantly different.

2+ 2+ 2.2. MitoTEMPO Regulation of [Zn ]i and Some Zn -Transporters in the Aged Left Ventricular Cardiomyocytes It has been already demonstrated that there is a close relationship between increases in oxidative 2+ stress, at most, ROS and [Zn ]i in cardiomyocytes under pathological condition [26,37,39], while the marked increase in ROS was demonstrated in the aged cardiomyocytes. Therefore, here, we 2+ first determined the cellular [Zn ]i in the aged ventricular cardiomyocytes isolated from 24-month 2+ old rats. As can be seen in Figure1A,B, the average level of [Zn ]i was significantly high (about 3-fold) in the aged cardiomyocytes loaded with FluoZin-3 compared to those of 6-month old rats. A mitochondria-targeting antioxidant MitoTEMPO treatment (1-µM for 5-h) of the cells provided 2+ marked recovery in this high [Zn ]i in the aged cardiomyocytes. To examine the regulation of some Zn2+-transporters such as ZIP7, ZIP8, ZnT7, and ZnT8, which were previously investigated in the ventricular cardiomyocytes under pathological conditions [37,39], we determined their protein expression levels in total cell homogenates using Western blot analysis. As shown in Figure1C,D, there were significantly increased ZIP7 (left in C) with decreased ZIP8 (right in C), whereas the ZnT7 level was found to decrease significantly (left in D) with increased ZnT8 (right in D). These changes could recover significantly a treatment of the aged cells with mitochondrial antioxidant MitoTEMPO. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 18 Int. J. Mol. Sci. 2019, 20, 3783 4 of 18

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0 .0 0 .0 0 0 .0 0 0 .0 2+ 2+ Figure 1.1. DeterminationDetermination of of intracellular intracellular free free zinc zinc ([Zn ([Zn]i2+)] levelsi) levels and and associated associated Zn Zntransporters2+ transporters in the in 2+ agedthe aged left ventricularleft ventricular cardiomyocytes. cardiomyocytes. (A) Representing (A) Representing confocal confocal images images of cardiomyocytes of cardiomyocytes and [Zn and]i 2+ measurement[Zn2+]i measurement protocol protocol in freshly in isolated freshly cardiomyocytes isolated cardiomyocytes loaded with loaded Zn -selective with Zn fluorescent2+-selective 2+ dyefluorescent FluoZin-3. dye Arrows FluoZin on-3. theArrows right on represent the right the repr Zn esent/Pyr the and Zn TPEN2+/Pyr applications, and TPEN applications, respectively. 2+ Therespectively. maximum The and maximum minimum and fluorescent minimum signals fluorescent obtained, signals as described obtained in, as the described methods in with the Znmethods/Pyr (5-µM) and TPEN (50-µM). (B) The bars representing the average [Zn2+] for all groups. The protein with Zn2+/Pyr (5-μM) and TPEN (50-μM). (B) The bars representing the averagei [Zn2+]i for all groups. expressionThe protein levels expression of ZIP7 levels and ZIP8 of ZIP7 (C, left and and ZIP8 right, (C respectively),, left and right and, respectively for ZnT7 and), and ZnT8 for ( DZnT7, left and right,ZnT8 respectively)(D, left and right in total, respectively cellular homogenates.) in total cellular Cardiomyocytes homogenates. were Cardiomyocytes incubated with were MitoTEMPO incubated µ µ (1withm MitoTEMPOfor 5 h). Scale ( bar1 µm in the for confocal 5 h). Scale image: bar 20 inm. the Numbers confocal of image used cardiomyocytes: 20 µm. Numbers isolated of fromused 5–6 rats/groups were >20 per fluorescence protocols. Data were presented as Mean SEM. The protein cardiomyocytes isolated from 5–6 rats/groups were >20 per fluorescence protocols.± Data were expression data were obtained with double assays in each sample from each group for each type of presented as Mean±SEM. The protein expression data were obtained with double assays in each measurement (numbers of rats/groups = 5–6) and Significance level accepted as * p < 0.05 vs. 6-month sample from each group for each type of measurement (numbers of rats/groups = 5–6) and old group, # p < 0.05 vs. 24-month old group. Significance level accepted as *p < 0.05 vs. 6-month old group, #p < 0.05 vs. 24-month old group. 2+ 2.3. Aging-Associated Redistribution of Cellular [Zn ]i in the Aged Cardiomyocytes To examine the regulation of some Zn2+-transporters such as ZIP7, ZIP8, ZnT7, and ZnT8, which 2+ wereTo previously examine the investigated redistribution in of the the ventricular cellular [Zn cardiomyocytes]i, we used a cell under model pathological to mimic the conditions aged cell by[37,39] using, we rat determined ventricular celltheir line protein H9c2, expression as used previously levels in [ 37total,39 ,42cell]. homogenates As can be seen using in Figure Western2A,B, blot we 2+ determinedanalysis. As the shown [Zn in]i levelFigure in H9c21(C and cells D with), there galactose were significantly treatment (D-gal, increased 50-µM, ZIP7 about (left 3-fold in C higher) with thandecreased that of ZIP8 nontreated (right in cells) C), whereas similar to the one ZnT7 obtained level inwas ventricular found to decrease cardiomyocytes significantly isolated (left freshly in D) fromwith 24-monthincreased oldZnT8 rats (right (about in D 3-fold). These higher changes than thatcould of recover 6-month sig oldnificantly rats given a treatment in Figure 1ofB). the aged 2+ cells Followingwith mitochondrial the validation antioxidant of the cellularMitoTEMPO. [Zn ]i in these D-GAL50 treated cells, we determined 2+ 2+ 2+ [Zn ] levels in both mitochondria and S(E)R, separately ([Zn ]Mit and [ Zn ]SER). As can be seen in 2+ Figure2.3. Aging2C,D,-Associated respectively, Redistr theibution [Zn of]Mit Cellularincreased [Zn2+ over]i in 2-foldthe Aged in theCardiomyocytes aged cardiomyocytes compared to 2+ those of 6-month old rats. However, although there was a slight decrease in [Zn ]SER level measured To examine the redistribution of the cellular [Zn2+]i, we used a cell model to mimic the aged cell in the aged group, the differences between the aged group and young group were not statistically by using rat ventricular cell line H9c2, as used previously [37,39,42]. As can be seen in Figure 2 (A significant. Furthermore, MitoTEMPO treatment (1-µM for 24-h) fully preserved the increased level of and B), we determined the [Zn2+]i level in H9c2 cells with galactose treatment (D-gal, 50-µM, about 3- [Zn2+] in the aged cardiomyocytes (Figure2C). fold higherMit than that of nontreated cells) similar to one obtained in ventricular cardiomyocytes

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Figure 2. Determination of intracellular and subcellular [Zn2+] levels in D-galactose induced aged Figure 2. Determination of intracellular and subcellular [Zn2+]i ilevels in D-galactose induced aged H9c2H9c2 cell cell line. line. (A (A) )r representativeepresentative confocal confocal images images of of H9 H9c2c2 cells cells incubated incubated with with reducing reducing sugar sugar D D galactose (D-Gal, 50 mg/mL for 48-h incubation) to monitor [Zn2+] ;(B) the measured [Zn2+] values galactose (D-Gal, 50 mg/mL for 48-h incubation) to monitor [Zn2+]I; (IB) the measured [Zn2+]i valuesi in H9c2in H9c2 cells cells with with different different concentrations concentrations of of(D (D-Gal-Gal (10 (10-,50-,100-,50-,100 mg/mL mg/mL for for 48 48-h)-h) ( (left).left). Effects Effects of of mitochondria-targeted antioxidant MitoTEMPO treatment, (MitoT; 1 µM for 24-h) on [Zn2+] in D-Gal mitochondria-targeted antioxidant MitoTEMPO treatment, (MitoT; 1 µM for 24-h) on [Zn2+]ii in D-Gal 2+ (50 mg/L) treated H9c2 cells (right); (C) representing graph to measure the [Zn 2+]Mit in a genetically (50 mg/L) treated H9c2 cells (right); (C) representing graph to measure the [Zn ]Mit in a genetically encoded FRET-based Zn2+ sensor (Mit-eCALWY4) in D-Gal treated H9c2 cells (left). The measurement encoded FRET-based Zn2+ sensor (Mit-eCALWY4) in D-Gal treated H9c2 cells (left). The measurement protocols are given in methods. The bar graph is representing the effect of the MitoTEMPO treatment protocols are given in methods. The bar graph is representing the effect of the MitoTEMPO treatment 2+ 2+ on [Zn ]mit level; (D) representing graph to measure the level of Zn in Sarco(endo)plasmic reticulum on [Zn2+]mit level; (D) representing graph to measure the level of Zn2+ in Sarco(endo)plasmic reticulum 2+ 2+ [Zn ]S(E)R in a genetically encoded FRET-based Zn sensor (ER-eCALWY6) (left) and effect of [Zn2+]S(E)R in a genetically encoded FRET-based Zn2+ sensor (ER-eCALWY6) (left) and effect of 2+ 2+ MitoTEMPO treatment on [Zn ]SER (right). Arrows (in C and D) represent the TPEN and Zn /Pyr MitoTEMPO treatment on [Zn2+]SER (right). Arrows (in C and D) represent the TPEN and Zn2+/Pyr applications, respectively. Numbers of used cardiomyocytes isolated from 5–6 rats/groups were >25 applications, respectively. Numbers of used cardiomyocytes isolated from 5–6 rats/groups were >25 per fluorescence protocols. Scale bar: 10-µm. Data were presented as Mean SEM. Data were presented per fluorescence protocols. Scale bar: 10-µm. Data were presented as± Mean±SEM. Data were as Mean SEM. Significance level accepted as * p < 0.05 vs. untreated H9c2 group, # p < 0.05 vs. D-Gal presented± as Mean±SEM. Significance level accepted as *p < 0.05 vs. untreated H9c2 group, #p < 0.05 treated H9c2 group. vs. D-Gal treated H9c2 group. 2.4. Confirmation of Mitochondrial Function and ROS Level in Aging-Modeled H9c2 Cells Following the validation of the cellular [Zn2+]i in these D-GAL50 treated cells, we determined To validate the aging in D-GAL50 treated H9c2 cells, we measured the mitochondrial membrane [Zn2+] levels in both mitochondria and S(E)R, separately ([Zn2+]Mit and[ Zn2+]SER). As can be seen in potential (MMP) and ROS level in these cells. As can be seen in Figure3A–D, respectively, MMP Figure 2 (C and D, respectively), the [Zn2+]Mit increased over 2-fold in the aged cardiomyocytes was found to be depolarized and the ROS level was significantly high in D-GAL50 treated H9c2 cells compared to those of 6-month old rats. However, although there was a slight decrease in [Zn2+]SER levelcompared measured to those in the of untreatedaged group, H9c2 the cells. differences between the aged group and young group were not statisticallyWe further significant. treated these Furthermore, D-GaL50 treated MitoTEMPO H9c2 cells treatment with MitoTEMPO (1-µM for (1-24µ-hM) fully for 24-h preserved incubation) the and then measured the MMP and ROS level. These parameters were found to be fully preserved with increased level of [Zn2+]Mit in the aged cardiomyocytes (Figure 2C). MitoTEMPO treatment. 2.4. Confirmation of Mitochondrial Function and ROS Level in Aging-Modeled H9c2 Cells To validate the aging in D-GAL50 treated H9c2 cells, we measured the mitochondrial membrane potential (MMP) and ROS level in these cells. As can be seen in Figure 3 (A to D, respectively), MMP was found to be depolarized and the ROS level was significantly high in D-GAL50 treated H9c2 cells compared to those of untreated H9c2 cells.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 18

We further treated these D-GaL50 treated H9c2 cells with MitoTEMPO (1-µM for 24-h Int. J. Mol. Sci. 2019, 20, 3783 6 of 18 incubation) and then measured the MMP and ROS level. These parameters were found to be fully preserved with MitoTEMPO treatment. (-) FCCP A B

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rofepresentative chloromethyl-2 confocal0,70-dichlorodihydrofluoroscein images of chloromethyl-2’,7’ diacetate-dichlorodihydrofluoroscein (DCFDA, 10-µM for 60-mindiacetate incubation) (DCFDA, 10loaded-µM for H9c2 60- cells.min incubation To monitor) loaded the ROS H9c2 level, cells. the To DCFDA monitor loaded the ROS cells level, were the calibrated DCFDA loaded with H cells2O2 (100-wereµ calibratedM). Results with are given H2O2 as(100 percentage-μM). Results changes are givenin the fluorescenceas percentage intensities; changes in (D the) estimated fluorescence ROS intensitieslevels of all; ( groups.D) estimated Scale ROS bar: 10-levelsµm. of Numbers all groups. of Scale used bar: cardiomyocytes 10-µm. Numbers isolated of used from cardiomyocytes 5–6 rats/groups wereisolated>25 from per fluorescence 5–6 rats/groups protocols. were Data >25 were per presented fluorescence as Mean protocols.SEM. SignificanceData were level presented accepted as ± Mean±SEM.as * p < 0.05 vs.Significance untreated level H9c2 accepted group, # pas< *0.05p < 0.05 vs. D-Gal vs. untreated treated H9c2 H9c2 group. group, #p < 0.05 vs. D-Gal treated H9c2 group. 2.5. Redistribution of Zn2+-Transporters among Suborganelles in the Aged Cardiomyocytes Isolated˙ from the Left Ventricle of 24-Month Old Rats 2.5. Redistribution of Zn2+-Transporters among Suborganelles in the Aged Cardiomyocytes İsolated from the Left VentricleWe first isolatedof 24-Mo eithernth Old mitochondria Rats or S(E)R and cytosolic fractions in the isolated left ventricular cardiomyocytes (Figure4A, Figure5A, respectively). Following the validation of these fractions We first isolated either mitochondria or S(E)R and cytosolic fractions in the isolated left with specific markers (COXIV for mitochondria and SERCA2a for S(E)R), we determined the protein ventricular cardiomyocytes (Figure 4A, Figure 5A, respectively). Following the validation of these expression levels of the Zn2+-transporters given in Section 2.2. The evaluation of these transporters in fractions with specific markers (COXIV for mitochondria and SERCA2a for S(E)R), we determined isolated S(E)R fraction showed that both ZIP7 (B) and ZIP8 (C) did not change in the mitochondria

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 18 Int. J. Mol. Sci. 2019, 20, 3783 7 of 18 the protein expression levels of the Zn2+-transporters given in Section 2.2. The evaluation of these transporters in isolated S(E)R fraction showed that both ZIP7 (B) and ZIP8 (C) did not change in the fraction of the aged cardiomyocytes compared to those of young cardiomyocytes. However, both ZnT7 mitochondria fraction of the aged cardiomyocytes compared to those of young cardiomyocytes. and ZnT8 did increase in these fractions signiﬁcantly (D and E, respectively). MitoTEMPO treatment However, both ZnT7 and ZnT8 did increase in these fractions significantly (D and E, respectively). (1-µM, for 5-h) of the aged cardiomyocytes fully preserved the changes in the expression levels of these MitoTEMPO treatment (1-µM, for 5-h) of the aged cardiomyocytes fully preserved the changes in the Zn2+-transporters measured in the mitochondria fraction. expression levels of these Zn2+-transporters measured in the mitochondria fraction. The ZIP7 level (with respect to the SERCA2a level) was increased signiﬁcantly in S(E)R fraction of The ZIP7 level (with respect to the SERCA2a level) was increased significantly in S(E)R fraction the aged cardiomyocytes isolated from the 24-month old rats compared to those of 6-month old rats of the aged cardiomyocytes isolated from the 24-month old rats compared to those of 6-month old (Figure5B) with no change in ZIP8 (C). Furthermore, the level of ZnT7 was markedly depressed in the rats (Figure 5B) with no change in ZIP8 (C). Furthermore, the level of ZnT7 was markedly depressed S(E)R fraction of these aged cells compared to those of young cells (D) with no change in ZnT8 level in the S(E)R fraction of these aged cells compared to those of young cells (D) with no change in ZnT8 (E). Furthermore, MitoTEMPO treatment the aged cardiomyocytes fully preserved the changes in the level (E). Furthermore, MitoTEMPO treatment the aged cardiomyocytes fully preserved the changes expression levels of these Zn2+-transporters measured in the S(E)R fraction. in the expression levels of these Zn2+-transporters measured in the S(E)R fraction.

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FigureFigure 5.5. ProteinProtein expressionexpression levelslevels ofof ZIP7,ZIP7, ZIP8,ZIP8, ZnT7,ZnT7, andand ZnT8ZnT8 inin isolatedisolated endoplasmicendoplasmic reticulumreticulum fractionfraction ofof thethe leftleft ventricularventricular cardiomyocytes.cardiomyocytes. ((A)) To validate proper isolation of Sarco(endo)plasmic,Sarco(endo)plasmic, (S(E)R),(S(E)R), andand cytosoliccytosolic fractions, SERCA2a SERCA2a ( (atat 110 110 kDa kDa)) was was used used as as S( S(E)RE)R marker, marker, while while GAPDH GAPDH (at (at37 37kDa kDa)) was was used used as a as cytosolic a cytosolic marker. marker. Aging Aging did didnot change not change the protein the protein expression expression levels levels of ZIP8 of ZIP8(C) and (C) ZnT8 and ZnT8 (E) in (E the) in isolated the isolated S(E)R S(E)R fraction fraction,, while while there there was wasa significant a significant increase increase in ZIP7 in ZIP7 (B) and (B) anddecrease decrease in ZnT in ZnT77 decreased decreased (D ()D measured) measured in in the the aged cardiomyocytes. cardiomyocytes. MitoTEMPOMitoTEMPO treatment treatment preservedpreserved allall thesethese changeschanges significantly.significantly. DataData werewere obtainedobtained withwith doubledouble assaysassays inin eacheach samplesample fromfrom each group for each type of measurement (numbers of rats/groups = 4–5) and were presented as each group for each type of measurement (numbers of rats/groups = 4–5) and were presented as Mean SEM. Significance level accepted as * p < 0.05 vs. 6-month old group, # p < 0.05 vs. 24-month Mean±SEM.± Significance level accepted as *p < 0.05 vs. 6-month old group, # p < 0.05 vs. 24-month old old group. group. 2.6. MitoTEMPO Preserves the Increases in Both Oxidation and K-Acetylation Levels in Left Ventricular Aged2.6. MitoTEMPO Cardiomyocytes Preserves the Increases in Both Oxidation and K-Acetylation Levels in Left Ventricular Aged Cardiomyocytes In order to detect the direct effect of aging on the heart via changes in oxidation status of the cells, we firstIn measuredorder to detect the levels the direct of total effect oxidant of aging status on (TOS) the andheart total via antioxidant changes instatus oxidation (TAS) status in the of aged the cardiomyocytescells, we first measured compared the to levels the young of total cardiomyocytes. oxidant status Figure (TOS6) Aand shows total that antioxidant the TOS levelstatus (left) (TAS was) in markedlythe aged cardiomyocytes high in the aged compared cardiomyocytes to the young compared cardiomyocytes. to those of Figure young 6A group, shows while that the the TASTOS levellevel (right)(left) was in the markedly age group high was in significantly the aged cardiomyocytes lower than those compared of the young to those group. of young MitoTEMPO group, treatmentwhile the (1-TASµM level for 5-h)(right of) thein the aged age cardiomyocytes group was significantly not fully low buter significantly than those of preserved the young these group. changes. MitoTEMPO treatmentTo demonstrate (1-µM for the 5-h eff) ectsof the of agingaged on cardiomyocytes the protein-thiol not oxidation fully but level significantly in isolated preserved left ventricular these cardiomyocytes,changes. we measured the total and oxidized protein-thiol levels in the aged cardiomyocytes comparedTo demonstrate to those of the young effects cardiomyocytes. of aging on the As protein can be-thiol seen oxidation in Figure level6B, aging in isolated induced left significantventricular increasescardiomyocytes in oxidized, we measured protein-thiol the total level and (right) oxidized with protein no change-thiol levels in total in protein-thiolthe aged cardiomyocytes level (left). MitoTEMPOcompared to those treatment of young of the cardiomyocytes. aged cardiomyocytes As can be fully seen preserved in Figure 6B, these aging changes induced measured significant in theincreases aged-group. in oxidized protein-thiol level (right) with no change in total protein-thiol level (left). MitoTEMPO treatment of the aged cardiomyocytes fully preserved these changes measured in the aged-group.

Int. J. Mol. Sci. 2019, 20, 3783 9 of 18

In the last part of our study, we determined the total K-Acetylation level in isolated left ventricular aged cardiomyocytes. Compared to those of the young group, the total K-Acetylation was signiﬁcantly high in the aged group (Figure6C). Further evaluation of the K-Acetylation level in isolated suborganelle fractions, the mitochondrial K-Acetylation (Figure6D) was markedly high in the aged cardiomyocytes compared to those of young cardiomyocytes, whereas there was no change in the S(E)R fraction K-Acetylation (Figure6E). MitoTEMPO (1- µM for 5-h) treatment of the aged cardiomyocytes fully preservedInt. J. Mol. Sci. these 2018, changes19, x FOR PEER to the REVIEW levels of the young cardiomyocytes. 9 of 18

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t i 0 0 M 0 .0 Figure 6. Determination of protein thiol oxidation level and protein K-Acetylation status in the aged Figure 6. Determination of protein thiol oxidation level and protein K-Acetylation status in the aged cardiomyocytes. (A) total antioxidant status and total oxidant status and MitoTEMPO effects on these cardiomyocytes. (A) total antioxidant status and total oxidant status and MitoTEMPO effects on these parameters measured in the aged cardiomyocytes; (B) protein thiol oxidation and MitoTEMPO effects parameters measured in the aged cardiomyocytes; (B) protein thiol oxidation and MitoTEMPO effects measured in the aged cardiomyocytes; (C) acetylated lysine (K-Acetylation) level measured with the measured in the aged cardiomyocytes; (C) acetylated lysine (K-Acetylation) level measured with the immune-blotting method in the aged cardiomyocytes (left), and (D) mitochondria-graded (middle) immune-blotting method in the aged cardiomyocytes (left), and mitochondria-graded (middle) and and (E) sarco(endo)plasmic-graded (right) fractions of the cardiomyocytes. Data were obtained with sarco(endo)plasmic-graded (right) fractions of the cardiomyocytes. Data were obtained with double double assays in each sample from each group for each type of measurement (numbers of rats/groups assays in each sample from each group for each type of measurement (numbers of rats/groups = 4–5) = 4–5) and were presented as Mean SEM. Significance level accepted as * p < 0.05 vs. 6-month old and were presented as Mean±SEM. Significance± level accepted as *p < 0.05 vs. 6-month old group, # p group, # p < 0.05 vs. 24-month old group. < 0.05 vs. 24-month old group. 3. Discussion In the last part of our study, we determined the total K-Acetylation level in isolated left 2+ ventricularThe overall aged cardiomyocytes. aim of the present Compared study was to those first of to the examine young thegroup, distribution the total K of-Acetylation cellular [Zn was] 2+ 2+ amongsignificantly suborganelles, high in the such aged as group S(E)R (Figure and mitochondria 6C). Further([Zn evaluat]SERionand of the [Zn K-Acetylation]Mit) in ventricular level in 2+ cardiomyocytesisolated suborganelle during fractions, aging in mammalians.the mitochondrial Second, K-Acetylation we examined (Figure whether 6D) some was Znmarkedly-transporters high in 2+ canthe redistributeaged cardiomyocytes among suborganelles compared to and those contribute of young to cardiomyocytes, redistributed of free whereas Zn among there was suborganelles no change 2+ inin these the S aged(E)R cardiomyocytes. fraction K-Acety Third,lation to (E understand). MitoTEMPO whether (1-µM [Zn for]Mit 5plays-h) treatment a predominant of the role aged to thecardiomyocytes alteration of fully aging-associated preserved these alterations changes in to cardiac the levels function of the by young using cardiomyocytes. a mitochondrial-targeting antioxidant, MitoTEMPO. Similar to previous data obtained in hyperglycemic cardiomyocytes [39,43], 2+ we3. Discussion monitored and quantified significantly high [Zn ]i in aged cardiomyocytes compared to those of

The overall aim of the present study was first to examine the distribution of cellular [Zn2+] among suborganelles, such as S(E)R and mitochondria ([Zn2+]SER and [Zn2+]Mit) in ventricular cardiomyocytes during aging in mammalians. Second, we examined whether some Zn2+-transporters can redistribute among suborganelles and contribute to redistributed of free Zn2+ among suborganelles in these aged cardiomyocytes. Third, to understand whether [Zn2+]Mit plays a predominant role to the alteration of aging-associated alterations in cardiac function by using a mitochondrial-targeting antioxidant, MitoTEMPO. Similar to previous data obtained in hyperglycemic cardiomyocytes [39,43], we monitored and quantified significantly high [Zn2+]i in aged cardiomyocytes compared to those of young cells by using confocal examinations. Furthermore, our present data also demonstrated that

Int. J. Mol. Sci. 2019, 20, 3783 10 of 18 young cells by using confocal examinations. Furthermore, our present data also demonstrated that 2+ there is a redistribution of cellular [Zn ]i among suborganelles under aging, ones similar to those of 2+ hyperglycemic cardiomyocytes [37,39]. These altered levels of either cellular [Zn ]i or redistribution of cellular compartments can likely be one of the potential mediators for the age-dependent development of cardiac dysfunction. Furthermore, our present results suggest that these Zn2+-carriers, such as ZIP7 and ZnT7 as well as ZIP8 and ZnT8, can carry Zn2+ in opposite directions across either S(E)R or mitochondria membranes, which are similar to ones previously demonstrated in hyperglycemic cardiomyocytes [37,39]. In addition, the activity of these transporters depends on modulations oxidation, phosphorylation, or K-Acetylation during aging in cardiomyocytes, and, therefore, these 2+ changes in those carries, in turn, contribute to that of redistribution of cellular [Zn ]i among 2+ compartments. In this regard, we previously have demonstrated that high cellular [Zn ]i could contribute to both electrical and mechanical dysfunction in left ventricular cardiomyocytes, in a manner of a close relationship between increased ROS and depressed cellular activity through high cellular 2+ 2+ 2+ [Zn ]i [39,43]. Importantly, it has been considered that [Zn ]i increases resulted in releases of Zn during cardiac-cycle [44], which can further trigger the production of higher pro-oxidants, leading to more oxidative damage in ventricular cardiomyocytes [43]. With a reversed relationship, oxidants in 2+ 2+ the cells can induce significant increases in [Zn ]i, in part, the release of Zn from metalloproteins [26], while we observed marked alterations in the heart function in hyperglycemic cardiomyocytes through 2+ 2+ increased oxidative stress and high [Zn ]i [45]. Evidently, it has been pointed out that not only [Zn ]i 2+ but also [Ca ]i was increased in left ventricular cardiomyocytes under oxidative stress to different 2+ extents [26]. Another important point related with increases in [Zn ]i (about 70% with much less 2+ increase in [Ca ]i in cardiomyocytes) under increased oxidative stress in mammalians such as diabetic rats, the parallelism between ionic changes and an unbalanced oxidant-status/antioxidant capacity [46]. 2+ More importantly, supporting our present data, authors demonstrated that elevations in [Ca ]i and 2+ 2+ 2+ [Ca ]mit were coupled with [Zn ]i and [Zn ]mit in cardiomyocytes under aldosteronism [47]. Zn2+ is a redox-inert ion in biology, and, therefore, it also plays important role in redox-status of cells while its levels are modulated by the redox-state of the cells such as ROS production [48], although Zn2+ has opposing effects in the cells including either elevate the antioxidant capacity or lead to ROS release at toxic levels [49]. Indeed, literature data documented that zinc-coordination with cysteine ligands can induce the sulfur-ligands oxidization [46,49], while there is a relationship between increased 2+ [Zn ]i and elevation of ROS in mammalian cells by inhibiting mitochondria [50,51]. Since it has been shown that oxidative phosphorylation rates and and ATPase-activities were markedly depressed in mitochondria from heart preparations under pathological conditions, including hyperglycemia [52], our similar findings in the present study strongly support the current hypothesis related with the 2+ 2+ important role of mitochondrial function via changes not only in [Zn ]i but also in [Zn ]Mit in heart dysfunction during aging with insulin resistance development. In addition, the high protein thiol oxidation and depressed antioxidant capacity in the aged cells (although aging is a physiological event) are in accordance with our previous finding with hyperglycemic cardiomyocytes. Indeed, we did 2+ present previously that the increased [Zn ]i was associated with high phosphorylation in cardiac Ca2+–release channels (RyR2) via phosphorylation of both protein kinase A (PKA) and CamKII [43]. 2+ In summary, it seems that the increased [Zn ]mit, with an important contribution to increases in both 2+ [Zn ]i and [Ca2+]i, under increased oxidative stress together with depressed antioxidant-defense in the cells under aging, can induce important alterations in cardiomyocyte function. Therefore, one 2+ can suggest that a disbalance in [Zn ]i in cardiomyocytes, under either pathological conditions or 2+ aging, can result in a disbalance in cellular signaling mechanisms via interference between the [Zn ]i 2+ signaling and the [Ca ]i signaling of cardiomyocytes. In the present study, we demonstrated that the total K-Acetylation measured in total cellular preparation was significantly high in the aged group compared to that of the young group. Furthermore, with a detailed examination in isolated organelle levels, the K-Acetylation level in isolated mitochondria fraction was significantly high, one similar to that of total cellular total acetylation; however, there were Int. J. Mol. Sci. 2019, 20, 3783 11 of 18 no differences in the S(E)R fraction K-Acetylation of these two groups. As known from previous studies, an altered mitochondrial function is an underlying basis for the increased sensitivity to oxidative stress in the aged heart, such as a decreased capacity to oxidize fatty acids and enhanced dependence on glucose metabolism [53,54]. Aging can impair the structure, and function of mitochondria and induces mitochondrial oxidative phosphorylation [6,54]. Our data are in line with what is already known in this field because protein K-Acetylation underlies the aging-associated cardiac diseases. Further data on increased ROS in the aged cardiomyocytes support the hypothesis on ROS-associated increase in protein acetylation [54]. Indeed, the authors showed a 58% increase in protein acetylation of mitochondria from 24-month old rat heart compared to those of 6-month old rats, while a ROS scavenger preserved all those changes. Previously, we demonstrated that the expression levels of ZIP7 and ZnT7 in left ventricular cardiomyocytes under hyperglycemia acted as mediators of ER stress as well as ER stress-associated cardiac dysfunction [37]. These two transporters are carrying Zn2+ in opposite directions across S(E)R membranes and they can phosphorylate under pathological conditions, though likely inducing important changes in S(E)R with deleterious consequences for cell function [37]. Although the levels of ZIP7 and ZnT7 in the S(E)R fraction of aged cardiomyocytes changed similar to those of the 2+ hyperglycemic cardiomyocytes [37], here, the change in [Zn ]i was not statistically significant between aged- and young-group cardiomyocytes. In addition, we, with a further study, did show that both ZIP7 and ZnT7 localized to both mitochondria and S(E)R and contribute to cellular Zn2+-muffling between cellular-compartments in either hyperglycemic or hypertrophic cardiomyocytes via affecting S(E)R-mitochondria coupling [39]. Supporting these previous data, here, we provided further information that mitochondria behave as essential mediators of the regulation of cellular during aging and provide an important contribution to aging-associated insufficient cardiac function and/or heart dysfunction. Our present data are supported with the previous data and with an already existing hypothesis for the presence of the same transporters localized to both S(E)R and mitochondria as well as to other compartments in the same cell being responsible from cellular [Zn2+] distribution in the cytosol and other organelles under physiological conditions [55]. Indeed, a differential cytosolic-localization of ZnT8 in β-cells has also previously been shown [56] in addition to a differential subcellular localization of the splice variants of ZnT5 in human intestinal cells [57]. Importantly, in the present study, we demonstrated that the mitochondrial levels of both ZnT7 and ZnT8 in the aged left ventricular cardiomyocytes were found to be markedly increased while ZIP7 and ZIP8 remainede 2+ unchanged, inducing a marked increase in [Zn ]Mit. These increases can further affect not only S(E)R-mitochondria coupling but also the increase of ROS production into cells. These changes further can contribute to the disruption of mitochondrial dynamism during the aging heart. Our present data with MitoTEMPO, importantly, provided very important information about the role of mitochondria as a target compartment of cardiomyocytes to the aging-associated alterations at the cellular level. Our overall data, therefore, showed, for the first time, that mitochondria are an important target for aging and mitochondria directed antioxidants like MitoTEMPO can provide substantially an important cardio-protective benefit to control of cellular and subcellular [Zn2+] in the aged cardiomyocytes via altered levels of some Zn2+-transporters (Figure7). Any therapy with mitochondria-targeted antioxidant will have an important impact as the cardioprotective approach for aging-associated cardiac dysfunction in mammalians. Int. J. Mol. Sci. 2019, 20, 3783 12 of 18 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 12 of 18

2+ 2+ FigureFigure 7. A 7. schematic A schematic representation representation to demonstrateto demonstrate the the re-distribution re-distribution of [Znof [Zn]2+i ]andi and [Zn [Zn2+]]MitMit with 2+ with re-distributionre-distribution of of Zn Zn2+-transporters-transporters in in the the aged aged cardiomyocytes cardiomyocytes (24-month (24-month old old rats) rats during) during the the developmentdevelopment of aging-associated of aging-associated cardiac cardiac dysfunction. dysfunction. Our dataOur ondata redistribution on redistribution of between of between cytosol, cytosol, 2+ 2+ 2+ mitochondriamitochondria and sarco(endo)plasmicand sarco(endo)plasmic reticulum reticulum S(E)R S ([Zn(E)R (][Zni, ([Zn2+]i, ([ZnMit,2+ ([ZnMit, ([Zn]SER2+]SER, respectively),, respectively), at 2+ at most,most, via via redistributed redistributed Zn Zn–transporters2+–transporters are are summarized summarized in the in aged the aged cardiomyocytes. cardiomyocytes. The close The close 2+ associationassociation between between increased increased ROS andROS [Zn and [Zn]i are2+] alsoi are consideredalso considered to be to an be important an important contributor contributor to to that ofthat insu offfi insufficientcient cardiac cardiac function function in old in individuals. old individuals. Here, Here,, , and, , andare  representing are representing an increase, an increase, 2+ ↑ ↓ ↔ decrease,decrease, and no and change no ch inange the in expression the expression levels levels of Zn of –transporters.Zn2+–transporters. 4. Material and Methods 4. Material and Methods 4.1. Experimental Animals 4.1. Experimental Animals Experiments were performed with 24-month old (24-month, aged group) and 6-month old (6-month,Experiments young group) were Wistar performed male rats. with Rats 24 were-month kept old in standard(24-month animal, aged housinggroup) and rooms 6-month and they old (6- had tapmo waternth, young and fed group) with standardWistar male chow rats. ad Rats libitum, were freely kept and in standard daily. The animal fasting housing blood glucose rooms leveland they and oralhad glucosetap water tolerance and fed test with was standard applied, chow as described ad libitum, previously freely [and58]. Alldaily experimental. The fasting procedures blood glucose werelevel performed and oral in accordanceglucose tolerance with the test standards was applied, of the as European described Community previously guidelines[58]. All experimental on the careprocedures and use of laboratory were perfor animals,med in and accordance were approved with by the the standards Ankara University of the European with a reference Community numberguidelines of 2016-18-165 on the care in accordance and use of with laboratory the guide animals for the, and care were and use approved of laboratory by the animals Ankara (dated:University 21.09.2016).with a reference number of 2016-18-165 in accordance with the guide for the care and use of laboratory animals (dated: 21.09.2016). 4.2. Fresh Cardiomyocyte Isolation˙ from the Left Ventricle 4.2. Fresh Cardiomyocyte İsolation from the Left Ventricle The hearts of all experimental animals, following being anesthetized by sodium pentobarbital (30 mg/kg,The i.p.), hearts were of perfused all experimental retrogradely animals, and performed following standard being anesthetized enzymatic digestion, by sodium as describedpentobarbital elsewhere(30 mg/kg, [59]. Briefly, i.p.), thewere cannulated perfused hearts retrogradely through the and coronary performed arteries standard on a Langendor enzymaticff apparatus digestion, as described elsewhere [59]. Briefly, the cannulated hearts through the coronary arteries on a Langendorff apparatus were perfused (3–5 min) with a Ca2+-free solution (in mM): 145 NaCl, 5 KCl,

Int. J. Mol. Sci. 2019, 20, 3783 13 of 18

2+ were perfused (3–5 min) with a Ca -free solution (in mM): 145 NaCl, 5 KCl, 1.2 MgSO4, 1.4 Na2HPO4, 0.4 NaH2PO4, 5 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), and 10 glucose at pH 7.4, bubbled with O2 at 37 ◦C and then followed the perfusion with a solution containing 1 mg/mL collagenase (Collagenase A, Boehringer) for 30–35 min. Left ventricular cardiomyocytes were isolated following the digestion with and the only the cells with rod-shaped, quiescent, and responses electric-ﬁeld stimulation were used for all experiments The percentage of viable cells was 70–80% in every heart following increases of Ca2+ in the medium to a concentration of 1-mM. To examine the role of a mitochondrial-targeting antioxidant on aged-cardiomyocytes, half of isolated cardiomyocytes from the 24-month old group was treated with MitoTEMPO (1-µM in the medium that cells were incubated) for 5-h before the experiments.

4.3. Cell Culturing The H9c2 cell-line was derived from embryonic rat heart and purchased from the American Type Culture Collection (CRL 1446) and grown at 37 ◦C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) in the presence of 5.5 mM glucose, as described previously [60]. Briefly, the cells were grown to a density of about 105 cells/cm2 and cultured as a monolayer in supplemented with 10% fetal calf serum, 50 U/mL penicillin-G and 50 µg/mL streptomycin in a humidified atmosphere at 37 ◦C. To obtain aged cells, the sub-confluent cells were treated with different concentration of reducing sugar D-galactose (D-GaL: 10-, 50-, and 100-mg/L) for 48-h to accelerate the glycation of macromolecules. Aging cells evaluated with a marked increase of cytosolic reactive oxygen species (ROS) with depolarized mitochondrial membrane potential (MMP) after the treatment. A group of D-Gal induced aged H9c2 cells were treated with 1-µM MitoTEMPO for 24-h.

4.4. Determination of Intracellular and Suborganelle Levels of Free Zn2+ by Confocal Microscopy Measurements

2+ 2+ Intracellular (cytosolic) free Zn level ([Zn ]i) was measured in freshly isolated cardiomyocytes by using confocal microscopy (Leica TCS SP5, Wuerzburg, Germany), as described previously [39]. 2+ 2+ 2+ To monitor Sarco(endo)plasmic reticulum and mitochondrial free Zn levels ([Zn ]SER and [Zn ]Mit), we used eCALWY sensors ER-eCALWY6 and Mit-eCALWY4, respectively. Image analysis was performed with ImageJ software (1.44p) using a homemade-macro, as described elsewhere [42]. To calculate free Zn2+, the maximum and minimum ﬂuorescence ratios, obtained with either a heavy-metal-chelator N,N,N’, N’-tetrakis(2-pyridylmethyl) ethylenediamine, TPEN (50-µM) or 2+ 2+ 2+ Zn -saturation with 100-µM ZnCl2 and Zn -ionophore pyrithione (Zn /Pyr, 5-µM) and calculated with their speciﬁc Kd values (ER-eCALWY6: Kd = 2.9 nM; Mito-eCALWY4: Kd = 60 pM) for all groups. A group of D-Gal induced aged H9c2 cells were treated with 1-µM MitoTEMPO for 24-h.

4.5. Confocal Imaging of Mitochondrial Membrane Potential and ROS Level The mitochondrial membrane potential, MMP, was measured in H9c2 cells using the fluorescence-based method similar to ones measured in freshly isolated cardiomyocytes, as described previously [61]. Briefly, cells were loaded with a membrane-permeant single wavelength fluorescence dye JC-1 (5-µM for 30-min) and imaged with a confocal fluorescence microscope (Leica TCS SP5). For calibration, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 5-µM) was used. Confocal imaging of ROS levels in H9c2 cells, similar to ones in freshly isolated cardiomyocytes as described previously [61], was performed and quantified. Briefly, cells were loaded with a ROS indicator chloromethyl-20,70-dichlorodihydrofluoroscein diacetate (DCFDA, 10-µM for 60-min) and then are examined with a laser scanning microscope (Leica TCS SP5). For maximal fluorescence intensity, cells were exposed to a HEPES-buffered solution supplemented with H2O2 (100-µM).

4.6. Isolation of Sarco(endo)Plasmic Reticulum and Mitochondria Fractions from Left Ventricular Cardiomyocytes The S(E)R-fractionation from isolated left ventricular cardiomyocytes was determined by using ER-isolation kit (Sigma, E0100), as described previously [37]. Briefly, cardiomyocytes were homogenized Int. J. Mol. Sci. 2019, 20, 3783 14 of 18 in isotonic extraction-buffer and then crude microsomal-fraction was isolated from post-mitochondrial fraction using ultracentrifugation. Western blot analysis was carried out using primary antibodies against ZIP7, ZIP8, ZnT7, and ZnT8. To confirm a proper S(E)R-isolation, SERCA2 (Santa Cruz, SC-8094) was used as S(E)R marker. Isolation of mitochondrial fraction was performed using a mitochondria isolation kit (Thermo, 89874, Paisley, UK) in isolated cardiomyocytes, according to the manufacturer’s instructions, as described previously [39]. Protein levels were measured by Bradford Assay (Pierce Biotechnology, Waltham, MA, USA) with bovine serum albumin as standard solution, and homogenates were loaded on to 12% sodium dodecyl sulphate (SDS) polyacrylamide gels. To confirm a proper mitochondria-isolation, COX IV (Abcam, ab1474, Cambridge, UK) was used as a mitochondrial marker.

4.7. Western Blotting Following incubations, cells were lysed in ice-cold Radioimmunoprecipitation assay buffer (RIPA, 50 µM Tris/HCl, pH at 7.4, 150 µM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (w/v) and %1 protease inhibitor cocktail) by vortexing for 1 min. Following lysis, the samples were centrifuged at 10,000 rpm for 5-min and the protein content of the supernatants was determined × using Bradford Assay (Pierce Biotechnology, Waltham, MA, USA) with bovine serum albumin as standard. Homogenates were loaded unto 8–10% SDS polyacrylamide gels and electrophoresed under constant voltage (150 V) for 2–3 h, and then electrically transferred onto polyvinylidene fluoride (PVDF) membrane for 30 min in the semi-dry transfer system. The membrane was blocked in 3–5% (wt/vol) albumin with 0.02% Tween-20 TBS buffer for 1 h and then incubated with recommended dilutions overnight with the primary antibodies; ZIP7 (Santa Cruz, sc-83858, 1:500, Dallas, TX, USA), ZIP8 (Protein Tech, 20459-1-AP, Manchester, UK) ZnT7 (Santa Cruz, sc-160948), ZnT8 (Santa Cruz, sc-98243), GAPDH (Santa Cruz, sc-365062), SERCA2 (Santa Cruz, sc-8094) purchased from Santa Cruz Biotechnology and COX4 (ab1474), from Abcam. The membranes were incubated with horseradish peroxidase-linked secondary antibodies and immunoreactivity was visualized using luminol-based chemiluminescence reaction. Immunoreactive bands on X-ray film were analyzed using open access ImageJ software (NIH) program. All reagents were obtained from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. All fluorescent dyes were purchased from Molecular Probes (Eugene, Oregon, USA). N,N,N0, N0-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) were obtained from Sigma (St. Louis, MO, USA). TPEN was prepared as a 1-mM stock solution in dimethyl sulfoxide (DMSO).

4.8. Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) Measurement in Cardiomyocytes TOS and TAS levels were measured in plasma as well as in cardiomyocytes by using the commercially available kit (RL0024, Rel Assay Diagnostics, Gaziantep, Turkey), as described previously [62]. Brieﬂy, we used the novel automated method, which is based on the bleaching of the characteristic color of a more stable ABTS (2,20-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation by antioxidants. The results are expressed as mmol Trolox equivalent/L. The TOS level in plasma was measured using commercially available kits (RL0024, Rel Assay Diagnostics, Gaziantep, Turkey) as described previously [62]. Shortly, the oxidation reaction is enhanced by glycerol molecules abundantly present in the reaction medium. The ferric ion produced a colored complex with xylenol orange in an acidic medium. The color intensity, measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with H2O2 and the results are expressed in terms of µMH2O2 equivalent/L.

4.9. Determination of Oxidized Protein Thiol Level in Isolated Cardiomyocytes Total and oxidized sulfhydryl (thiol) groups were estimated with Ellman’s reagent, as described previously [27]. Brieﬂy, cardiomyocytes were thawed and lysed in 0.2 M Tris/HCl buﬀer pH at 8.1 containing 2% Na-dodecyl sulfate. For the measurement of total thiol-groups in cell homogenates, Int. J. Mol. Sci. 2019, 20, 3783 15 of 18 both for reaction mix and background reaction mix, cell lysate and 20% trichloroacetic acid mixtures were centrifuged for 10-min at 13,000 rpm. Following the centrifugation, the supernatants were collected and added NaOH. Both for total and oxidized thiol-group measurements, 2-mM 5,50-dithiobis-(2-nitrobenzoic acid) was added to reaction mixtures and let them to stand for 20-min. Then, all reaction mixtures and background reaction mixtures were put in 96-well plates and absorbances were read using microplate-reader (SpectraMax Plus384, Molecular Devices LLC, San Jose, CA, USA) at 412-nm.

4.10. Determination of Protein K-Acetylation in Cardiomyocytes Acetylation of lysine residues within proteins has emerged as an important mechanism used by cells to overcome many physiological processes. Assessment of lysine acetylation of proteins was performed according to the immune-blotting assay. As described above, cells were lysed, centrifuged and the protein content of the supernatants was determined [39]. Homogenates were electrophoresed and transferred onto polyvinylidene ﬂuoride (PVDF) membrane in the semi-dry transfer system. The membrane was incubated with recommended dilutions overnight with the Anti-Acetyl lysin antibody (Abcam; ab21623). This antibody detects several bands including 11 kDa, 15 kDa, 45 kDa, and 50 kDa sizes. We analyzed a range between 30- and 50 kDa band sizes to correlate K-Acetylation of Zn-transporters of experimental groups.

4.11. Data Analysis and Statistics All data were presented as a mean ( SEM) and a two-tailed Student’s t-test was used to determine ± statistical signiﬁcance (p < 0.05).

5. Conclusions

2+ In the present study, we have, for the first time, demonstrated that [Zn ]i-homeostasis and its 2+ 2+ suborganelle levels such as [Zn ]Mit and [Zn ]SER, are altered in left ventricular cardiomyocytes from 24-month old rats compared with those of 6-month old rats. Furthermore, we also demonstrated that these above alterations are due to not only changes in the total protein expression levels of the Zn2+-transporters such as ZIP7, ZIP8, ZnT7, and ZnT8 but also due to the re-distribution of these Zn2+-transporters in mitochondria and S(E)R. Moreover, our data, for the first time, have shown that the cellular changes in the parameters of ROS, both total and mitochondrial lysine acetylation, and 2+ protein-thiol oxidation are contributing to the changes in cellular [Zn ]i-homeostasis in the aged cardiomyocytes. More importantly, confirming our hypothesis, a mitochondrial-targeting antioxidant, 2+ MitoTEMPO provided important cardioprotection via positively affecting the [Zn ]i-dyshomeostasis of the ventricular cardiomyocytes from 24-month old rats.

Author Contributions: B.T. designed and supervised the research and provided the final approval of the version to be published; Y.O. and E.T. contributed and performed the experiments and analyzed the data. Funding: This study was supported through a grant from TUBITAK SBAG-216S979. Conflicts of Interest: The authors declare no conflict of interest.

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