An Investigation on Molecular Basis of the Effects of SGLT2 İnhibitor Dapagliflozin on Hyperglycemia-Associated Heart Dysfunction

Abstract

Objectives: It has been reported that sodium glucose co-transporter (SGLT) inhibitors lowering blood glucose in diabetic patients via inhibiting glucose re-absorption. Although it has not been clearly identified the effects of these inhibitors on heart function, these inhibitors may act like Na+/H+-exchanger (NHE) inhibitors. In this study, we were aimed to clarify the underlying cellular mechanism of a SGLT2-inhibitor, Dapagliflozin (DAPA), on hyperglycemic embryonic rat ventricular cells (H9c2 cell line) via electrophysiological measurements. Materials and Methods: One group of H9c2 cells were incubated with high glucose (25 mM) medium for 24-hours and 48-hours at 37 °C to obtain hyperglycemic (HG) cardiomyocytes. Another group of H9c2 cells were incubated together with high glucose medium and DAPA (D185360, TORONTO Research Chemicals; 100 nM or 1 μM). Intracellular ion concentrations, ([X]in), reactive oxygen species, ([ROS]in) and mitochondrial membrane potential, (MMP) were monitored via specific fluorescent dyes (DCFDA, FluoZin-3AM, JC-1, SNARF, FURA-2AM and SBFI) with confocal microscope and microspectrofluorometer. All data are presented as mean (± SEM) and statistical analysis performed by student t-test. Significance level considered at p<0.05 for all comparisons. Results: Due to the toxic effects of 1 μM DAPA incubation, all experiments were conducted with 100 nM DAPA incubated cells. [Na+]in levels were not significantly changed in any group. Moreover, increased [H+]in level in HG incubation (48-hours) significantly augmented following DAPA treatment (24-hours and 48-hours). However [Zn+2]in levels in HG incubated (24-hours and 48-hours) cells were significantly increased, DAPA treatment had no further effect. In addition, increased [Ca+2]in in HG reduced with DAPA treatment to control values. Importantly, DAPA treatment significantly reduced elevated [ROS]in but did not improve depolarized MMP in HG cardiomyocytes. Conclusion: In conclusion these results indicate that DAPA treatment restores [H+]in and [ROS]in homeostasis and improve contraction-relaxation activity of the heart in HG conditions.

Keywords

• Hyperglycemia
• Sodium-Glucose Co-Transporter Inhibitors
• Oxidative Stress
• Heart Function
• Intracellular Ionic-Homeostasis

SGLT2 İnhibitörü Dapagliflozinin Hiperglisemi-Aracılı Kalp Fonksiyon Bozukluğu Üzerindeki Etkisinin Moleküler Temellerinin İncelenmesi

Abstract

Amaç: Sodyum glukoz ko-transporter (SGLT) inhibitörlerinin diyabetli bireylerde glukoz re-absorbsiyonunu inhibe ederek kan şekerini düşürdüğü ileri sürülmektedir. Bu inhibitörlerin kalp fonksiyonu üzerindeki etkileri henüz tam olarak bilinmemesine karşın, Na+/H+-değiştokuşçusu (NHE) inhibitörü gibi etkiler gösterebildiği ileri sürülmektedir. Bu çalışmada, SGLT2-inhibitörü Dapagliflozin (DAPA), in vitro olarak sıçan embriyonik hiperglisemik ventriküler hücrelerine (H9c2-hücre hattı) uygulanarak hücre seviyesindeki etki mekanizmasının elektrofizyolojik yöntemler kullanılarak incelenmesi hedeflenmiştir. Gereç ve Yöntem: H9c2 hücreleri deney koşullarına hazır hale getirildikten sonra, H9c2’ler bir grubu 25 mM glukoz ile 24-saat veya 48-saat 37 °C’de inkübe edilerek hiperglisemik kardiyomiyositler (HG) elde edilmiş, bir diğer grubun inkübasyonu ise 25 mM glukoz ve DAPA (D185360, TORONTO Research Chemicals; 100 nM veya 1 μM) ile aynı sürelerde gerçekleştirilmiştir. Hücreiçi iyon derişimleri ([X]in, [ROS]in ve mitokondri membran potansiyeli, (MMP, X= Na+, Ca2+, H+, Zn2+) ölçümleri hücreler özel floresans boyalarla yüklenerek (DCFDA, FluoZin-3AM, JC-1, SNARF-1AM, FURA-2AM ve SBFI) konfokal mikroskobu ve mikrospektrofluorometre kullanılarak ölçülmüştür. Gruplar arası karşılaştırmalar student t-testi ile gerçekleştirilmiş ve anlamlılık (p) değeri 0,05’den küçük değerler kabul edilmiştir. Bulgular: Normal ve hiperglisemik hücrelerde 1 μM DAPA uygulamasında toksik etkiler gözlendiğinden, tüm incelemeler 100 nM DAPA uygulaması ile yapılmıştır. [Na+]in değeri bu uygulamalarda değişmezken, HG inkübasyonda (48-saat) istatistiksel olarak anlamlı düzeyde artış olan [H+]in değeri, DAPA uygulaması ile (24-saat ve 48-saat) çok belirgin olarak artmıştır. Buna karşın, HG’li hücrelerde (24-saat ve 48-saat) artmış olan [Zn+2]in değerlerini DAPA uygulaması etkilememiştir. Buna karşın, HG’li hücrelerde (24-saat ve 48-saat) artmış olan [Ca+2]in değerlerinin DAPA uygulaması ile normal değerlere yakın olduğu gözlenmiştir. Bunlara ek olarak, HG koşullarında önemli derecelerde artmış olan [ROS]in değerinin DAPA ile normal değerler civarında gözlendiği, fakat HG sonucu depolarize durumdaki MMP’lerini etkilemediği sonuçları elde edilmiştir. Sonuç: Sonuç olarak elde edilen verilerimiz, SGLT2 inhibitörlerinin HG hücrelerde, artan [H+]in ve [ROS]in baskılayarak, artmış olan bazal [Ca+2]in’nin düzelmesine ve böylece kalbin kasılma-gevşeme aktivitesinin HG koşullarında normal fonksiyon göstermesine yol açabileceğini işaret etmektedir

Keywords

• Hiperglisemi
• Sodyum-Glukoz Ko-Taşıyıcı İnhibitörleri
• Oksidatif Stres
• Kalp Fonksiyonu
• Hücre İçi İyon-Homeostazları

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