Browsing by Author "Olgar, Yusuf"

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    Increased free Zn2+ correlates induction of sarco(endo)plasmic reticulum stress via altered expression levels of Zn2+‐transporters in heart failure
    (2018) Olgar, Yusuf; Tıp Fakültesi; Durak, Ayşegül; Tuncay, Erkan; Bitirim, Ceylan Verda; Özçınar, Evren; İnan, Mustafa Bahadır; Akar, Ahmet Ruchan; Akçalı ,Kamil Can
    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 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.
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    Metabolik sendromlu sıçan kortikal nöronlarında endoplazmik retikulum stresiyle ilgili faktörlerin araştırılması
    (Ankara Üniversitesi Bilimsel Araştırma Projesi, 2018) Can, Belgin; Kızıl, Şule; Göktürk, Hilal; Bayram, Pınar; Billur, Deniz; Turan, Belma; Olgar, Yusuf; Toy Durak, Ayşegül; Tıp Fakültesi
    Metabolik sendromlu sıçan kortikal nöronlarında endoplazmik retikulum stresiyle ilgili faktörlerin araştırılması Metabolik sendrom (MetS), insülin direnci temelinde ortaya çıkan ve klinik tablosunda glukoz intoleransı veya tip 2 diyabet, abdominal obezite, hipertansiyon, hiperlipidemi ile karakterize; proinflamatuvar ve protrombotik öğeleri bulunduran ve prematüre aterosklerozun yer aldığı artmış kardiyovasküler hastalık riski oluşturan bir patolojidir. MetS’in en önemli parametrelerinden birisi insulin direncidir. Endoplazmik retikulumun (ER) normal fonksiyonunun bozulması sonucu ER stresi ortaya çıkmaktadır. Son yıllarda yapılan çalışmalar insülin sinyal yolağının düzenlenmesinde ER stresinin önemli bir rol oynadığını göstermektedir. ER stresinin metabolik hastalıklar, nörodejeneratif hastalıklar ve kardiyovasküler hastalıklar gibi çeşitli patolojik koşulları tetiklediği bilinmektedir. Bu nedenle çalışmamızda, MetS modeli oluşturulmuş sıçanların kortikal nöronlarında ER stresinin varlığını ve egzojen insülinin ER stresi üzerine etkilerini göstermek üzere ER stresiyle ilişkili belirteçlerin araştırılması amaçlanmıştır. Çalışmamızda 3 grup içinde toplam 30 adet 2 aylık Wistar albino erkek sıçan kullanılmıştır. Kontrol grubunda yer alan sıçanlar (n=10) standart sıçan yemi ve çeşme suyu ile beslenirken, MetS (n=10) ve MetS+İnsülin (n=10) grupları standart sıçan yemine ek olarak %32 sükroz içeren çeşme suyu ile 20 hafta boyunca beslenmiştir. 20 haftalık süre sonucunda MetS ve MetS+İnsülin gruplarında MetS deney modelinin oluşumu bozulmuş glukoz toleransı, insülin direnci, arteriyel kan basıncında artış ve serum insülin seviyesinde artış sonucunda doğrulanmıştır. MetS deney modelinin oluşumu doğrulandıktan sonra MetS+İnsülin grubundaki deney hayvanlarına 15 gün boyunca 3 IU/gün dozunda subkutan insülin uygulanmıştır. Bu sürenin sonunda deney hayvanları sakrifiye edilerek beyinleri çıkarılmıştır. Çıkarılan beyinlerde sol hemisfere ait prefrontal korteks bölgesi Western blotlama deneylerinde kullanılırken sağ hemisfere ait prefrontal korteks bölgesi ışık mikroskobik incelemeler ve ince yapı incelemeleri için kullanılmıştır. Deney hayvanlarından elde edilen beyin dokularında ER stres belirteçlerinden IRE1α, pIRE1, PERK, p-PERK, ATF6, GRP78, GSK3α/β ve pro-Caspase 12 ile apopitoz belirteçlerinden Caspase 3 ve TUNEL için incelemeler gerçekleştirilmiştir. İncelemeler sonucunda elde edilen veriler IBM SPSS 22 programı kullanılarak istatistiksel olarak değerlendirilmiştir. Hematoksilin-Eozin ve Krezil Viyole boyamaları ile gerçekleştirilen genel doku incelemelerinde gruplar arasında belirgin farklılıklar görülmemiştir. İmmünohistokimyasal işaretlemeler ve Western blotlama deneylerinden elde edilen bulgularda kontrol grubu ile karşılaştırıldığında MetS ve MetS+İnsülin gruplarında ATF6 ekspresyonundaki azalma ile EDEM, p-IRE1, GRP78 ve Caspase 3 protein ekspresyonlarındaki artış istatistiksel olarak anlamlı bulunmuştur. İnce yapı düzeyinde kontrol grubu ve MetS+İns grubu ile karşılaştırıldığında MetS grubunda nükleusun daha heterokromatik olduğu ve ER lümeninin genişlemiş olduğu görülmüştür. Bu çalışmanın sonuçları, kortikal nöronlarda ER stresi ve MetS arasındaki ilişkinin ortaya konulması açısından önemli bilgiler sunmaktadır
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    Mitochondria-Targeting Antioxidant Provides Cardioprotection through Regulation of Cytosolic and Mitochondrial Zn 2+ Levels with Re-Distribution of Zn 2+-Transporters in Aged Rat Cardiomyocytes
    (2019) Olgar, Yusuf; Tıp Fakültesi
    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 µM, 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.
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    Mitochondria-Targeting Antioxidant Provides Cardioprotection through Regulation of Cytosolic and Mitochondrial Zn 2+ Levels with Re-Distribution of Zn 2+-Transporters in Aged Rat Cardiomyocytes
    (2019) Olgar, Yusuf; Tıp Fakültesi
    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 µM, 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.