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. 2020 Apr 9;21(7):2608.
doi: 10.3390/ijms21072608.

Diallyl Trisulfide (DATS) Suppresses AGE-Induced Cardiomyocyte Apoptosis by Targeting ROS-Mediated PKCδ Activation

Dennis Jine-Yuan Hsieh  1   2 , Shang-Chuan Ng  3 , Ren-You Zeng  3 , Viswanadha Vijaya Padma  4 , Chih-Yang Huang  5   6   7   8   9 , Wei-Wen Kuo  3
Affiliations

Diallyl Trisulfide (DATS) Suppresses AGE-Induced Cardiomyocyte Apoptosis by Targeting ROS-Mediated PKCδ Activation

Dennis Jine-Yuan Hsieh et al. Int J Mol Sci. .

Abstract

Chronic high-glucose exposure results in the production of advanced glycation end-products (AGEs) leading to reactive oxygen species (ROS) generation, which contributes to the development of diabetic cardiomyopathy. PKCδ activation leading to ROS production and mitochondrial dysfunction involved in AGE-induced cardiomyocyte apoptosis was reported in our previous study. Diallyl trisulfide (DATS) is a natural cytoprotective compound under various stress conditions. In this study, the cardioprotective effect of DATS against rat streptozotocin (STZ)-induced diabetic mellitus (DM) and AGE-induced H9c2 cardiomyoblast cell/neonatal rat ventricular myocyte (NRVM) damage was assessed. We observed that DATS treatment led to a dose-dependent increase in cell viability and decreased levels of ROS, inhibition of PKCδ activation, and recuded apoptosis-related proteins. Most importantly, DATS reduced PKCδ mitochondrial translocation induced by AGE. However, apoptosis was not inhibited by DATS in cells transfected with PKCδ-wild type (WT). Inhibition of PKCδ by PKCδ-kinase-deficient (KD) or rottlerin not only inhibited cardiac PKCδ activation but also attenuated cardiac cell apoptosis. Interestingly, overexpression of PKCδ-WT plasmids reversed the inhibitory effects of DATS on PKCδ activation and apoptosis in cardiac cells exposed to AGE, indicating that DATS may inhibit AGE-induced apoptosis by downregulating PKCδ activation. Similar results were observed in AGE-induced NRVM cells and STZ-treated DM rats following DATS administration. Taken together, our results suggested that DATS reduced AGE-induced cardiomyocyte apoptosis by eliminating ROS and downstream PKCδ signaling, suggesting that DATS has potential in diabetic cardiomyopathy (DCM) treatment.

Keywords: AGE; DATS; PKCδ; apoptosis; cardiomyocyte.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Advanced glycation end-product (AGE) induced cardiac PKCδ protein expression, phosphorylation, and apoptosis in a dose- and time-dependent manner. H9c2 cells were treated (A) with different doses of AGE (50, 100, 150, 200, and 250 μg/mL) for 24 h and (B) for different time periods (0, 3, 6, 12, 24, and 36 h) with AGE at 250 μg/mL. Protein levels were analyzed by Western blotting and β-actin was used as a loading control.
Figure 2
Figure 2
Inhibitory effect of diallyl trisulfide (DATS) on AGE-induced cardiac apoptosis and PKCδ mitochondrial translocation. (A) Cells were treated with DATS at different concentrations (0, 1, 3, 5, 7, 10, 15, and 20 µM) for 24 h. Cell viability was examined using an MTT assay. (B) Western blotting was performed to check the effects of DATS on the expression of the survival marker p-AKT. (C) Inhibition of AGE-induced apoptosis was examined using an MTT assay to assess cell viability following DATS treatment. (D,E) Following treatment with DATS (5 and 10 µM) after 1 h, AGE (250 µg/mL) was added to induce apoptosis for 24 h. The levels of p-PKCδ protein and apoptosis-related markers were assessed by Western blotting. p-PKCδ expression levels in the cytosol and mitochondria of H9c2 cardiomyoblast cells were seperated using a mitochondria and cytosol separation kit and analyzed by Western blotting. β-actin and COX IV were used as a loading control. Values shown are means ± SD. Quantification of the results is shown (n = 3); * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control cells; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus AGE-treated cells.
Figure 3
Figure 3
Antioxidant effect of DATS on AGE-induced cardiac reactive oxygen species (ROS) generation. Following treatment with DATS (1, 5, and 10 μM) after 1 h, AGE (250 μg/mL) was added to induce ROS generation for 24 h. (A) 2’,7’-dichlorofluorescin diacetate (DCF-DA) and (B) MitoSOXTM Red Mitochondrial Superoxide Indicator were used to detect intracellular and mitochondrial ROS production. (C) Mitochondrial membrane potential (MMP) was determined via JC-1 staining. Values shown are means ± SD. Quantification of the results is shown (n = 3); *** p < 0.001 versus control cells; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus AGE-treated cells.
Figure 4
Figure 4
Cardiac PKCδ-dependent apoptosis induced by AGE is inhibited by selective kinase-deficient PKCδ (GFP-PKCδ-KD). (A) Cells were transfected with GFP-PKCδ-wild type (WT) at the indicated doses. (B) H9c2 cells were exposed to AGE (250 µg/mL) and transfected with GFP-PKCδ-KD at the indicated doses. Protein levels were analyzed by Western blotting and β-actin was used as a loading control.
Figure 5
Figure 5
DATS suppresses cardiac apoptosis by inhibiting PKCδ activation and its downstream apoptosis-related proteins following AGE exposure. (A) Cells were exposed to AGE (250 μg/mL) followed by DATS (10 µΜ) treatment or PKCδ-KD (2 μg) transfection. (B) Cells were transfected with PKCδ-WT plasmid followed by DATS or rott treatment. (C) NRVM or (D) H9c2 cells were exposed to AGE (250 μg/mL) with or without PKCδ-KD transfection or treatment with DATS in the presence or absence of PKCδ-WT transfection. Protein levels were analyzed by Western blotting and β-actin and GAPDH was used as loading controls. Values shown are means ± SD. Quantification of the results is shown (n = 3); * p < 0.05, ** p < 0.01, and *** p < 0.001 versus AGE + DATS-treated cells.
Figure 6
Figure 6
DATS suppresses cardiac apoptosis by inhibiting the activation of PKCδ and apoptosis-related signaling pathways in streptozotocin (STZ)-induced diabetic rats. (A) The schematic procedure of STZ-induced diabetes mellitus (DM) and DATS treatment. (B) Cardiac expression of phosphorylated PKCδ was examined by immunohistochemistry analysis. (C) Western blot analysis of the phosphorylation levels of PKCδ and apoptosis-related proteins in STZ-induced diabetic rat hearts. Values shown are means ± SD. Quantification of the results is shown (n = 3); * p < 0.05, ** p < 0.01 and *** p < 0.001 versus control and ## p < 0.01 versus DM.
Figure 7
Figure 7
A proposed pathway of DATS attenuating AGE-induced cardiomyocytes apoptosis by inhibiting ROS-mediated PKCδ activation.
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