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. 2018 Aug 1;9(4):647-663.
doi: 10.14336/AD.2017.0924. eCollection 2018 Aug.

Pkcδ Activation is Involved in ROS-Mediated Mitochondrial Dysfunction and Apoptosis in Cardiomyocytes Exposed to Advanced Glycation End Products (Ages)

Yao-Chih Yang  1 , Cheng-Yen Tsai  2   3 , Chien-Lin Chen  4 , Chia-Hua Kuo  5   6 , Chien-Wen Hou  5 , Shi-Yann Cheng  7   8   9 , Ritu Aneja  10 , Chih-Yang Huang #  11 , Wei-Wen Kuo #  1
Affiliations

Pkcδ Activation is Involved in ROS-Mediated Mitochondrial Dysfunction and Apoptosis in Cardiomyocytes Exposed to Advanced Glycation End Products (Ages)

Yao-Chih Yang et al. Aging Dis. .

Abstract

Diabetic patients exhibit serum AGE accumulation, which is associated with reactive oxygen species (ROS) production and diabetic cardiomyopathy. ROS-induced PKCδ activation is linked to mitochondrial dysfunction in human cells. However, the role of PKCδ in cardiac and mitochondrial dysfunction caused by AGE in diabetes is still unclear. AGE-BSA-treated cardiac cells showed dose- and time-dependent cell apoptosis, ROS generation, and selective PKCδ activation, which were reversed by NAC and rotenone. Similar tendency was also observed in diabetic and obese animal hearts. Furthermore, enhanced apoptosis and reduced survival signaling by AGE-BSA or PKCδ-WT transfection were reversed by kinase-deficient (KD) of PKCδ transfection or PKCδ inhibitor, respectively, indicating that AGE-BSA-induced cardiomyocyte death is PKCδ-dependent. Increased levels of mitochondrial mass as well as mitochondrial fission by AGE-BSA or PKCδ activator were reduced by rottlerin, siPKCδ or KD transfection, indicating that the AGE-BSA-induced mitochondrial damage is PKCδ-dependent. Using super-resolution microscopy, we confirmed that PKCδ colocalized with mitochondria. Interestingly, the mitochondrial functional analysis by Seahorse XF-24 flux analyzer showed similar results. Our findings indicated that cardiac PKCδ activation mediates AGE-BSA-induced cardiomyocyte apoptosis via ROS production and may play a key role in the development of cardiac mitochondrial dysfunction in rats with diabetes and obesity.

Keywords: advanced glycation end products (AGEs); apoptosis; diabetes mellitus (DM); mitochondrial; protein kinase C (PKC)δ; reactive oxygen species (ROS).

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Conflict of interest statement

Competing Interests The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.. AGE-BSA reduces cell viability, enhanced apoptosis and ROS generation in H9c2 cells in a time- and dose-dependent manner
(A) Cells were treated with 300 μg/ml of AGE-BSA for different time periods as indicated or non-glycated BSA. Cell viability was determined using MTT assays. (B) Cells were treated with AGE-BSA at different concentrations as indicated. Levels of apoptosis-related proteins were analyzed by western blotting. These are cropped blots; full-length blots are presented in Suppl. Figure S1. (C, D) Bar graphs show relative optical densities of the (fig. 1B) apoptosis and survival protein levels at 24 h. (E) Intracellular ROS levels of fluorescence intensities of DCF and (F) the mitochondrial ROS levels of AGE-BSA-exposed cardiac cells at the indicated doses and time periods were examined by flow cytometry. NAC (500 μM); rotenone (Rote) (0.1 μM). Bars indicate the mean ± SEM obtained from experiments performed in triplicate. *P<0.05, **P <0.01 and ***P<0.001 compared with the control group; #P<0.05 and ##P<0.01compared with the 300 μg/mg group; †P<0.05 when compared with 24 h or 36 h.
Figure 2.
Figure 2.. Among PKC isoforms, AGE-BSA specifically induces PKCδ protein expression and phosphorylation in cardiac cells
(A) Cells were treated with different doses of AGE-BSA with or without ROS scavengers (NAC) and mitochondrial complex I inhibitor (Rote) as indicated for 24 h. Western blot analyses were performed with antibodies against the PKC isoforms. These are cropped blots; full-length blots are presented in Suppl. Figure S2. (B) The densitometry measurements show the quantitative results of the western blots. Bars indicate the mean ± SEM obtained from experiments performed in triplicate. *P<0.05 and ***P<0.001 compared with the control group; ###P<0.001 compared with the AGE-BSA (300 μg/ml) group.
Figure 3.
Figure 3.. Cardiac PKCδ expression and phosphorylation as well as apoptosis-related proteins are increased in diseased animal models with elevated circulatory AGE levels
Western blot analysis of the cardiac expression and phosphorylation levels of PKC isoforms and apoptosis-related proteins in rats with (A) diabetes mellitus (DM) and (B) a high-fat (HF) diet. Serum AGE levels in rats with (C) DM; (D) HF diet. These are cropped blots; full-length blots are presented in Suppl. Figure S3. Protocols for animal models with DM and HF diets and serum AGE analysis were described in the methods section. (E) Cardiac expression of phosphorylated PKCδ was examined by immunohistochemistry analysis. Bars indicate the mean ± SEM obtained from experiments performed in triplicate. *P<0.05 and ***P<0.001 compared with the control group. DM, Diabetes mellitus; HF, high-fat diet.
Figure 4.
Figure 4.. AGE-BSA-induced cardiomyocyte apoptosis is mediated through PKCδ activation.
(A) The diagram depicts the domain organization of GFP-PKCδ. The GFP-PKCδ derivatives, including the wild-type (WT) and the kinase-deficient mutant (KD; K376R). Cells were treated with AGE-BSA (300 μg/ml) and (B) rottlerin (1-5 μM) or (C) PKCδ silencing. (D) Cells were transfected with GFP-fused PKCδ (GFP PKCδ-WT) at different doses as indicated and with (E) 1 μg rottlerin (1-5 μM). (F&G) H9c2 cells or (H) neonatal rat ventricular myocytes (NRVM) were exposed to AGEs (300 μg/ml) with or without (GFP PKCδ-KD) transfection or transfected with GFP PKCδ-WT in the presence of rottlerin (3 μM) or not. These are cropped blots, full-length blots of PKCδ and pPKCδ are presented in Suppl. Figure S4. SC, scramble; WT, wild type; KD, kinase-deficient; All the proteins were analyzed by western blotting using β-actin as a loading control.
Figure 5.
Figure 5.. AGE-BSA-induced cardiomyocyte apoptosis is mediated through ROS-dependent PKCδ activation
NRVM and H9c2 cells were exposed to AGE-BSA (300 μg/ml) for 24 h. Cells were co-treated with bryostatin 1, a PKCδ activator (100 nM), rottlerin, a PKCδ inhibitor (3 μM), NAC (500 μM), Rote (0.1 μM), Apo (10 μM) or siPKCδ (1 μg). (A) Expression and phosphorylation of PKCδ and (B, C, D) apoptosis-related proteins were examined by western blot analyses. These are cropped blots, full-length blots of PKCδ and pPKCδ are presented in Suppl. Figure S5. β-Actin was used as a loading control. N-acetylcysteine, NAC; Rote, rotenone; APO, apocynin; SC, scramble.
Figure 6.
Figure 6.. AGE-BSA-induced mitochondria damage and decreased biological function is through PKCδ activation and colocalization in cardiac cells
(A, B) Cells were treated with the PKCδ activator, bryostatin 1 (100 nM) or inhibitor, rottlerin, (3 μM) for 24 h following exposure to AGE-BSA (300 μg/ml). (C) AGE-BSA-exposed cells were transfected with PKCδ siRNA (1 μg) for 24 h. (D) Cells were transfected with GFP-PKCδ-WT plus rottlerin or AGE-BSA-exposed cells were transfected with GFP-PKCδ-KD. Mitochondrial damage was evaluated by mitochondrial mass, which was analyzed by flow cytometry. (E) AGE-BSA-treated cells were administered the inhibitor rottlerin (3 μM) or siPKCδ for 24 h. The mitochondrial fission and colocalization with PKCδ were examined by immune-fluorescence and analyzed by confocal microscopy. (F) Super resolution images of mitochondria structure in cells were analyzed with similar experimental procedures. Blue, DAPI (4, 6-diamidino-2-phenylindole)-stained nuclei; red, MitoTracker Red CMXRos-stained mitochondria. (G) The levels of proteins associated with mitochondrial fission and mitophagy in cells were analyzed with similar experimental procedure. These are cropped blots; full-length blots are presented in Suppl. Figure S6. Neonatal rat ventricular myocyte (NRVM) and H9c2 were then analyzed for (H) Cells were treated with AGE-BSA for 24?hr and then transfected with the PKCδ siRNA (1 μg) for 24?hr. Cell lysates were immunoprecipitated using antibodies against pPKCδT505/507. Protein expression was detected by immunoblotting. (I) The cytosolic and mitochondrial fractions were isolated and subjected to immunoprecipitation followed by western blot analysis. These are cropped blots; full-length blots are presented in Suppl. Figure S7. (J) Cellular oxygen consumption rate (O.C.R) and extracellular acidification (E.C.A.R) using an XF24 bioenergetics assay (Seahorse Bioscience, Billerica, MA). Data are expressed as the mean ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.001 compared with the control or GFP group, #P<0.05, ##P<0.01, ###P<0.001 compared with the AGE-BSA (300 μg/mg) group, †P<0.05 compared with the Bry1-treated group, ††P<0.01, †††P<0.001 compared with the GFP-PKCδ-WT overexpression group.
Figure 7.
Figure 7.
Molecular events of ROS-dependent PKCδ activation involved in the AGE-BSA-induced cardiomyocyte apoptosis. PKCδ activation is involved in the regulation of AGE-BSA-induced cell apoptosis via ROS production and may play a key role in the development of cardiac mitochondrial dysfunction in rats with diabetes or obesity.
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