- Email: [email protected]
Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging
IJCA-24023; No of Pages 8 International Journal of Cardiology xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging Zhimin Zha a,1, Junhong Wang a,b,1, Xiangming Wang a, Miao Lu a, Yan Guo a,c,⁎ a b c
Department of Gerontology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of Cardiology, Shengze Hospital of Jiangsu Province, Suzhou, China
a r t i c l e
i n f o
Article history: Received 4 September 2016 Accepted 6 November 2016 Available online xxxx Keywords: Advanced glycation end products Cardiac senescence Mitophagy Receptor for advanced glycation end products PINK1
a b s t r a c t Context and objectives: Advanced glycation end products (AGEs) can induce senescence in cardiomyocytes. However, its underlying molecular mechanisms remain unknown. Methods: Neonatal rat cardiomyocytes were incubated with AGEs, and cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-β-gal) activity and aging-associated p16 expression. In addition, mitophagic activity was evaluated by measuring the expression of the PINK1, Parkin, LC3 and p62 proteins. The mitophagy inhibitor cyclosporine A (CsA) or PINK1 siRNAs was then administered to cardiomyocytes to study the role of mitophagy in AGE-induced aging. Results: A significantly increased number of SA-β-gal positive cells and increased p16 protein levels were observed in cardiomyocytes treated with AGEs. Moreover, AGEs significantly increased the protein levels of PINK1 and Parkin as well as the LC3-II/LC3-I ratio, which occurred in a dose-dependent manner. However, the expression of p62 decreased significantly in the AGE group compared to the control. Surprisingly, both CsA and the knockdown of PINK1 by small-interfering RNA (siRNA) significantly decreased the LC3-II/LC3-I ratio and the PINK1 and Parkin protein levels in AGE-treated cardiomyocytes. Moreover, CsA treatment or knockdown of PINK1 expression attenuated the increased number of SA-β-gal positive cells and the upregulated p16 level in cardiomyocytes induced by AGEs. Conclusions: PINK1/Parkin-mediated mitophagy is involved in the process of cardiomyocyte senescence induced by AGEs, and a reduction in mitophagic activity might be a promising approach to block the senescent state in cardiomyocytes. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Aging, which is a process driven by randomly occurring damage and its accumulation, is an inevitable physiological phenomenon [1]. Ageassociated degenerative diseases such as cardiovascular diseases play a central role as the dominant cause of death due to the continuously increasing number of aged people. Cardiac aging is an independent risk factor for cardiovascular diseases and is associated with cardiac hypertrophy, diastolic dysfunction and compromised myocardial performance [2,3]. Many different hypotheses on the causes of aging have been proposed, including oxidative stress, mitochondrial dysfunction, telomere shortening, inflammation, and glycation, but the underlying mechanisms remain largely unknown.
⁎ Corresponding author at: Department of Gerontology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. E-mail address: [email protected] (Y. Guo). 1 These two authors contributed equally to this work.
Advanced glycation end products (AGEs) are a heterogeneous group of compounds that are formed by the non-enzymatic glycation of proteins, lipids and nucleic acids [1,4]. The accumulation of AGEs is one of the major mechanisms of aging and plays an important role in age-related diseases [5–8]. In the myocardium, the AGE-dependent modification of protein structure and related inhibition of function induce collagen cross-linking in the extracellular matrix and, subsequently, fibrotic cardiac remodeling, which leads to ventricular stiffness and diseases [9,10]. In addition, soluble AGEs can bind to specific cell surface receptors (RAGE being the best known) to activate several intracellular signaling pathways [11–13]. This is also associated with AGE-induced aging [12,14]. The aging process is coupled with another cell process, mitochondrial autophagy, which selectively targets and removes damaged or old mitochondria [15,16]. Mitophagy is reported to be an important process for establishing senescence [17,18]. Several studies have shown that changes in the mitophagy system are implicated in a plethora of agerelated diseases, such as Alzheimer's, Parkinson's and Huntington's diseases [19–21]. Recent observations have suggested that senescence
http://dx.doi.org/10.1016/j.ijcard.2016.11.161 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
2
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
and mitophagy share a number of common characteristics and are regulated by overlapping signaling pathways, which include the generation of reactive oxygen species, the induction of p53 and p21, and the dephosphorylation of retinoblastoma protein [22,23]. However, the potential roles of mitophagy during senescence remain unclear. The PINK1/Parkin pathway is a canonical mechanism that is involved in the regulation of mitophagy in mammal cells, and it was reported to be a mechanism in neurodegenerative disorders [24–26]. Further, accumulating evidence has indicated that AGEs can trigger mitophagy in a variety of cell types but that suppressing one of its receptors, RAGE, markedly reduces the formation of autolysosomes [27–29]. However, the effect of AGEs-activated mitophagy on AGEsinduced aging and the manner in which the mitophagic pathway changes are rarely reported in cardiomyocytes. For these reasons, we investigated the role of PINK1/Parkin-mediated mitophagy in AGEinduced senescence in cardiomyocytes. 2. Materials and methods 2.1. Drugs and reagents AGE-modified bovine serum albumin (AGE-BSA) was purchased from Calbiochem (Temecula, CA, USA). A neutralizing anti-RAGE antibody was purchased from R&D systems (Minneapolis, MN, USA). Cyclosporin A (CsA) was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies against PINK1, Parkin, p62 and p16 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-LC3 antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated goat antirabbit antibody was purchased from Abcam (Cambridge, MA, USA). The senescence-βGalactosidase staining kit was from the Beyotime Institute of Biotechnology (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were from Grand Island Biological Company (Gibco, Grand Island, NY, USA). The protein quantification assay kit was purchased from Bio-Rad (Hercules, CA, USA). 2.2. Cell culture and treatment The animal handling and use protocols complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No.85-23, revised 1996) and were approved by the Animal Care and Use Committee of Nanjing Medical University. Ventricular myocytes were isolated from neonatal SpragueDawley rats (1 to 3 days old) as described previously [30]. Hearts were removed quickly and washed three times with cold phosphate buffered saline (PBS). The left heart tissues were minced with scissors into 1 mm3 fragments and were incubated in collagenase II solutions for further digestion. The homogenized tissue was allowed to adhere to the 100mm dish in culture media (DMEM) containing 10% FBS for 2 h at 37 °C. Fibroblasts were separated by adhesion onto the dish during this process. The non-adherent cells, neonatal rat ventricular cardiomyocytes, were plated in a culture plate and cultured in DMEM medium containing 10% (v/v) FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The medium was replaced every 48 h. After 3 days of culture, when the cells reached 80% confluence, the cardiomyocytes could be used in the subsequent experiments. Cardiomyocytes were treated with different concentrations of AGEs (50, 100, 200 μg/ml) for 48 h to investigate the effects of AGEs on cardiomyocytes. Then, the cardiomyocytes were incubated with AGEs (100 μg/ml), RAGE antibody (2 μg/ml) or CsA (0.5 μmol/l), as specifically indicated, and cultured for 48 h. 2.3. Beta-galactosidase activity Senescence-associated beta-galactosidase (SA-β-gal) activity was measured using the β-gal staining kit at pH 6.0 following the manufacturer's protocol (Beyotime Institute of Biotechnology, Wuhan, China). Briefly, cells were seeded into six-well plates, cultured with the reagents as mentioned above for 48 h, fixed for 15 min at room temperature with 1 mL of fixative solution and then washed three times with phosphate-buffered saline. Next, the cells were incubated overnight at 37 °C with a staining solution mix containing X-gal. After being rinsed with PBS, cells were observed for the development of the blue coloration with a microscope at a magnification of ×400. 2.4. siRNA interference and gene transfection The siRNA oligonucleotides targeting PINK1 were proven to be effective by other researchers [31] and synthesized by GenePharma (GenePharma, Shanghai, China). The sequences were as follows: negative control siRNA (NC): 5´-GAU CAU ACG UGC GAU CAG ATT-3′ and PINK1 siRNA: 5′ -GCU GCA AUG CCG CUG UGU ATT-3′ . siRNAs were then transfected into cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Lipofectamine 3000 and siRNA were diluted in Opti-MEM medium. After briefly vortexing, the mixture was incubated at room temperature for 10– 15 min to allow the formation of siRNA-Lipofectamine 3000 complexes and then added to the cells in DMEM medium containing serum/antibiotic. After incubation for 48 h, the cells were used for the following experiments.
2.5. Western blot analysis For western blot, cells were lysed with ice-cold Cell Lysis Reagent containing protease and phosphatase inhibitors (BiYun Tian, China, P0013B) to extract total proteins. After the protein samples were heat-denatured, equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes by electrophoresis. After transfer, the membranes were blocked with 5% non-fat milk and incubated overnight with primary antibodies against PINK1, Parkin, p62, LC3 and p16. Membranes were subsequently washed in Tris-buffered saline plus Tween-20 (TBST) and incubated with anti-rabbit HRP-conjugated secondary antibodies for two hours at 4 °C. After washing the membrane in TBST three times, the protein levels were detected by chemiluminescence using an enhanced chemiluminescence (ECL) detection kit (Millipore Corporation, Temecula, CA, USA). The level of GAPDH was used as an internal control. 2.6. Statistical analysis All data were obtained from at least 3 independent experiments. The results were presented as the mean ± SD. Statistical analyses were carried out using the GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). One-way ANOVA was used to compare the difference in those groups. Statistical significance was determined at a P value of b0.05.
3. Results 3.1. AGEs induces cellular senescence in neonatal rat cardiomyocytes To investigate whether cardiomyocytes treated with AGEs showed a senescent phenotype, neonatal rat cardiomyocytes were treated with different concentrations of AGEs. The frequency of SA-β-gal-positive staining and the expression level of the senescence marker, p16, were investigated to evaluate the senescence level of the cell population. As shown in Figs. 3 and 5, 48 h of incubation with AGEs resulted in significantincreases in both the cellular SA-β-gal activity and p16 levels compared to the control group, suggesting that AGEs can directly induce senescence in cardiomyocytes. 3.2. Increased mitophagy in AGEs-treated cardiomyocytes To evaluate mitophagic activity in AGE-induced senescent cardiomyocytes, mitophagy was investigated by analyzing the levels of mitophagy-related proteins. The conversion of cellular protein LC3-I to LC3-II and the level of p62 are two critical hallmarks of the mitophagy process. As shown in Fig. 1A and B, an accumulation of LC3-II and a decreased level of P62 were observed in AGEs group compared to the control, indicating elevated mitophagic activity in AGE-induced senescent cardiomyocytes. Furthermore, this increase occurred in a dosedependent manner with increasing concentration of AGEs, and the peak value was observed when the concentration of AGEs was 200 μg/ml. Two other important mitophagy-associated proteins, PINK1 and Parkin, were also investigated by western blotting. Senescent cardiomyocytes exhibited significantly increased expression levels of PINK1 and Parkin compared to the control group (Fig. 1C and D). These results suggest that AGE-induced senescent cardiomyocytes have high mitophagic activity. 3.3. RAGE-mediated, AGEs-induced senescence and mitophagy in cardiomyocytes In the AGEs group, the percentage of SA-β-gal-positive cardiomyocytes and the expression of p16 were significantly higher than those in the normal control group. However, the RAGE antibody significantly decreased both the SA-β-gal activity and p16 level in the AGEs group (Fig. 3A and B). After cardiomyocytes were pretreated with anti-RAGE for 2 h, the LC3-II/LC3-I ratio and the PINK1, Parkin and p62 levels were determined by western blot. Significant decreases in the LC3-II/LC3-I ratio and in the PINK1 and Parkin levels were observed in comparison to the AGEs group. In addition, the expression of p62 was increased significantly in the AGEs + RAGE antibody group (Fig. 2A-D).
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
3.4. Inhibition of mitophagy alleviates senescence in AGE-treated cardiomyocytes To study the role of mitophagy in AGE-induced cardiomyocyte senescence, the cardiomyocytes were incubated with a mitophagy inhibitor (0.5 μmol/l CsA) [32,33] and AGEs. Our results revealed that the LC3-II/LC3-I ratio was significantly reduced and that the p62 level was markedly increased when CsA was administered to the AGEinduced senescent cardiomyocytes for two days. (Fig. 2A and B). These
3
data may indicate that mitophagy was involved in the process of AGEinduced cardiomyocyte senescence. In addition, the senescence status of CsA treated cardiomyocytes was further analyzed. The rate of SA-β-gal-positive staining was significantly reduced after the cells were coincubated with CsA (Fig. 3A). Consistent with effect on SA-β-gal, the expression level of p16 protein was also reduced in the CsA group (Fig. 3B). This indicated that mitophagy is involved in the process of AGE-induced cardiomyocytes senescence.
Fig. 1. AGEs induce mitophagy in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to increasing concentrations of AGEs (0–200 μg/ml) for 48 h. LC3 (A), p62 (B), PINK1 (C) and Parkin (D) levels were measured by western blotting. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
4
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
3.5. Mitophagy-accelerated senescence in cardiomyocytes is dependent on PINK1/Parkin activation Because the PINK1/Parkin signaling pathway plays a pivotal role in mitophagy, the protein levels of PINK1 and Parkin were analyzed in AGE-induced senescent cardiomyocytes. Our results demonstrated
that the PINK1 and Parkin levels were significantly increased compared to those of the control group (Fig. 2C and D). Next, the gene silencing of PINK1 was used to study the role of the PINK/Parkin pathway in AGEinduced senescent cardiomyocytes. Our results showed that the decreased PINK1 expression in PINK1 siRNA group significantly decreased the LC3-II/LC3-I ratio compared to the AGEs group (Fig. 4A and B).
Fig. 2. RAGE antibody and CsA both inhibit AGEs-induced mitophagy in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to 100 μg/ml AGEs alone or in combination with 2 μg/ ml RAGE antibody or 0.5 μmol/L CsA. LC3 (A), p62 (B), PINK1 (C) and Parkin (D) levels were measured by western blotting. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control. $$ P b 0.01 compared with the corresponding AGEs treatment alone.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
Consequently, the senescence status of the AGEs-treated cardiomyocytes was further studied in the cells that were coincubated with the PINK1 siRNA. Significant decreases in the percentage of SA-βgal-positive cells and the expression level of the p16 protein were observed in the PINK1 siRNA-treated group (PINK1 siRNA + AGEs) compared to cells in the control AGEs group (Fig. 5A and B). Collectively, our data revealed that PINK1/Parkin pathway-mediated mitophagy might play a pivotal role in AGE-induced cardiomyocyte senescence. 4. Discussion AGEs are responsible for degenerative diseases such as cardiovascular diseases due to their accumulation within molecules, cells and tissues during aging [34–37]. Glycation modifies the structure and function of proteins and induces an increase in stiffness and a reduction in elasticity in vessels and heart via crosslinking in the extracellular matrix [38–40], which suggests that AGEs are closely associated with cardiac aging. Our previous study demonstrated that D-galactose, a reducing
5
sugar that is involved in the formation of AGEs, could induce cardiomyocyte senescence [41]. In our study, AGEs increased both the cellular SAβ-gal activity and the p16 level, which are the most widely used biomarkers for cardiomyocyte senescence. SA-β-gal is an early marker of cellular senescence because it reflects the function of the lysosomes and appears as the result of their increased biogenesis [42]. The p16 protein, a cyclin-dependent kinase (CDK) 4/6 inhibitor [43], functions in the restriction of G1/S cell cycle progression and in the induction of senescence. Consequently, our results may indicate that AGEs play an important role in the process of cardiac aging. In addition, RAGE, a specific receptor of AGEs, is expressed in various cell types, including cardiomyocytes. The binding of AGEs with RAGE receptors could influence cardiac function by activating multiple processes, including inflammatory responses, oxidative stress, cardiac fibrosis and impaired calcium metabolism in the cardiovascular system [4,44,45]. In the present study, the inhibition of RAGE with a RAGE-antibody reduced both the SA-β-gal activity and p16 level during AGE exposure, indicating that RAGE mediated the AGE-elicited senescence of cardiomyocytes.
Fig. 3. RAGE antibody and CsA attenuate AGEs-induced senescence in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to 100 μg/ml AGEs alone or in combination with 2 μg/ml RAGE antibody or 0.5 μmol/l CsA. (A) SA-β-gal assay. Representative image of SA-β-gal positive cells (×400). SA-β-gal positive cells are stained in blue. (B) The expression level of p16 was determined by western blot. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control. $$ P b 0.01 compared with the corresponding AGEs treatment alone. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
6
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
Fig. 4. Downregulation of PINK1 attenuates the mitophagy mediated by AGEs in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to 100 μg/ml AGEs after transfection with negative control siRNA or PINK1 siRNA. PINK1 (A) and LC3 (B) levels were measured by western blotting. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control. $$ P b 0.01 compared with the corresponding AGEs treatment alone.
These results are consistent with previous studies [4,14]. Together, these data demonstrated that the AGEs-RAGE axis plays an important role in the process of cardiomyocyte senescence. Mitophagy plays a crucial role in maintaining cardiac homeostasis [46,47]. There are multiple stages, structures, and proteins involved in this process [16]. In our study, we found that the LC3-II/LC3-I ratio was upregulated and that the level of p62 was downregulated during the process of AGE-induced senescence in cardiomyocytes. An increase in LC3-II protein is considered a marker for elevated autophagosome formation, and a decrease in p62 protein is a marker for increased autophagic turnover [48]. Accordingly, our results indicated that mitophagy might be involved in the cardiomyocyte senescence induced by AGEs. Mitophagy and senescence are considered to be two distinct cellular events; however, changes in the expression of mitophagic markers have been observed in different senescent cell types, which indicates that these two events are closely related [49,50]. Our findings showed that inhibiting mitophagy activation via a mitophagy inhibitor, CsA [51,52], contributed to the reduced SA-β-gal activity and reduced p16 expression in AGEs-treated cardiomyocytes. These results indicated that mitophagy might facilitate the development of cardiac aging induced by AGEs. These results also show that inhibition of mitophagy alleviates the AGE-induced senescent state in cardiomyocytes, which is in line with previous data on other cell types [17,22,49,53]. However, a number of studies support an inverse association between mitophagy and senescence, showing that mitophagic activity decreased during aging [15,54,55]. It is as though mitophagy plays a Janus-type role in that they can be either cytoprotective or cytotoxic [56–58]. This discrepancy may result from differences in the experimental and clinical conditions (different triggers, cellular and microenvironmental conditions) and the autophagic extent. It is possible that autophagy, under baseline conditions, is required to repair mild damage. In this context, autophagy is a homeostatic mechanism for maintaining cellular function and survival. However, in other circumstances, e.g., specific
disease settings, excess levels of autophagy facilitate the loss of cellular functions, including cell senescence [53,59]. There are also numerous pieces of evidence suggesting that autophagy is now considered a death execution mechanism (i.e., type II programmed cell death) [60–63]. The abnormal mitophagy observed in previous studies is consistent with this hypothesis [64]. Taken together, we assumed that excessive mitophagy might impair cardiac function, which could ultimately result in cardiac aging when the cardiomyocytes were exposed to AGEs. PINK1 and Parkin activation is a canonical mechanism for mitophagy regulation in most mammals [24–26]. Upon mitochondrial damage, PINK1 globally accumulates on depolarized mitochondria and recruits Parkin from the cytosol to the mitochondria, which is part of an ubiquitylation-related process for the selective removal of mitochondria. Thus, we hypothesized that the PINK1/Parkin pathway might be involved in regulating AGEs-induce cardiomyocyte senescence. Interestingly, our results demonstrated that the depletion of PINK1 in cardiomyocytes resulted in a remarkable attenuation both in mitophagy activity and in cardiomyocyte senescence induced by AGEs. These findings might indicate that PINK1/Parkin mediated-mitophagy may be involved in cardiac aging induced by AGEs. Therefore, our results may indicate that PINK1/Parkin mediated mitophagy plays an important role in AGEs-induced senescence of cardiomyocytes. The current study has some limitations. First, we only studied the PINK1/Parkin signaling pathway, which is not the only regulator of mitophagy. We did not look at other signal pathways that can regulate mitophagy in this study. More importantly, additional experiments on animals or humans should be done, and more work should be focused in the future on the specific effects of mitophagy on cardiac aging. However, taken together, the present study, for the first time, has revealed that AGEs-induced mitophagy promotes senescence in cardiomyocytes via RAGE and activation of the PINK1/Parkin pathway. Our current work provides a new insight into the understanding of mitophagy and aging
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
7
Fig. 5. PINK1 silencing alleviates the senescent state of cardiomyocytes treated with AGEs. Neonatal rat cardiomyocytes were exposed to 100 μg/ml AGEs after negative control siRNA or PINK1 siRNA transfection. (A) SA-β-gal assay. Representative micrographs depict morphology of SA-β-gal positive cells (×400). SA-β-gal positive cells are stained in blue. (B) The expression level of p16 was determined by western blot. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control. $$ P b 0.01 compared with the corresponding AGEs treatment alone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in cardiomyocytes. Moreover, these findings may provide a potential strategy for regulating mitophagy appropriately, that is, in a way that may counteract cardiac aging. Conflict of interest statement The authors declare no potential conflicts of interest were disclosed. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC 81570328, Wang Junhong) and the “Sixth-Peak Talent” of Jiangsu Province (2011WSN-029 to Prof. Guo Yan and 2013WSN-036 to Dr. Wang Junhong). Prof. Guo Yan was also support by the Health Department of Jiangsu Province (z201301). References [1] A. Simm, B. Muller, N. Nass, et al., Protein glycation - between tissue aging and protection, Exp. Gerontol. 68 (Aug 2015) 71–75. [2] R.A. Boon, K. Iekushi, S. Lechner, et al., MicroRNA-34a regulates cardiac ageing and function, Nature 495 (7439) (Mar 7 2013) 107–110.
[3] Y.A. Chiao, MicroRNA-34a: a new piece in the cardiac aging puzzle, Circ. Cardiovasc. Genet. 6 (4) (Aug 2013) 437–438. [4] M. Fang, J. Wang, S. Li, Y. Guo, Advanced glycation end-products accelerate the cardiac aging process through the receptor for advanced glycation end-products/ transforming growth factor-beta-Smad signaling pathway in cardiac fibroblasts, Geriatr. Gerontol. Int. 16 (4) (Apr 2016) 522–527. [5] A. Simm, Protein glycation during aging and in cardiovascular disease, J. Proteome 92 (Oct 30 2013) 248–259. [6] L. Robert, J. Labat-Robert, Role of the Maillard reaction in aging and age-related diseases. Studies at the cellular-molecular level, Clin. Chem. Lab. Med. 52 (1) (Jan 1 2014) 5–10. [7] M. Cardenas-Leon, E. Diaz-Diaz, R. Arguelles-Medina, P. Sanchez-Canales, V. DiazSanchez, F. Larrea, Glycation and protein crosslinking in the diabetes and ageing pathogenesis, Rev. Investig. Clin. 61 (6) (Nov-Dec 2009) 505–520. [8] N. Nass, B. Bartling, A. Navarrete Santos, et al., Advanced glycation end products, diabetes and ageing, Z. Gerontol. Geriatr. 40 (5) (Oct 2007) 349–356. [9] S.Y. Choi, H.J. Chang, S.I. Choi, et al., Long-term exercise training attenuates agerelated diastolic dysfunction: association of myocardial collagen cross-linking, J. Korean Med. Sci. 24 (1) (Feb 2009) 32–39. [10] S. Willemsen, J.W. Hartog, Y.M. Hummel, et al., Tissue advanced glycation end products are associated with diastolic function and aerobic exercise capacity in diabetic heart failure patients, Eur. J. Heart Fail. 13 (1) (Jan 2011) 76–82. [11] K.H. Gaens, C.D. Stehouwer, C.G. Schalkwijk, Advanced glycation endproducts and its receptor for advanced glycation endproducts in obesity, Curr. Opin. Lipidol. 24 (1) (Feb 2013) 4–11. [12] C. Ott, K. Jacobs, E. Haucke, A. Navarrete Santos, T. Grune, A. Simm, Role of advanced glycation end products in cellular signaling, Redox Biol. 2 (2014) 411–429.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
8
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
[13] A.L. Tan, J.M. Forbes, M.E. Cooper, AGE, RAGE, and ROS in diabetic nephropathy, Semin. Nephrol. 27 (2) (Mar 2007) 130–143. [14] J. Liu, K. Huang, G.Y. Cai, et al., Receptor for advanced glycation end-products promotes premature senescence of proximal tubular epithelial cells via activation of endoplasmic reticulum stress-dependent p21 signaling, Cell. Signal. 26 (1) (Jan 2014) 110–121. [15] J.J. Lemasters, Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging, Rejuvenation Res. 8 (1) (Spring 2005) 3–5. [16] A. Hamacher-Brady, N.R. Brady, Mitophagy programs: mechanisms and physiological implications of mitochondrial targeting by autophagy, Cell. Mol. Life Sci. 73 (4) (Feb 2016) 775–795. [17] M. Narita, A.R. Young, M. Narita, Autophagy facilitates oncogene-induced senescence, Autophagy 5 (7) (Oct 2009) 1046–1047. [18] A. Shaik, A. Schiavi, N. Ventura, Mitochondrial autophagy promotes healthy aging, Cell Cycle 15 (14) (2016 Jul 17) 1805–1806. [19] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (1) (Jan 11 2008) 27–42. [20] A. Perl, mTOR activation is a biomarker and a central pathway to autoimmune disorders, cancer, obesity, and aging, Ann. N. Y. Acad. Sci. 1346 (1) (Jun 2015) 33–44. [21] S. Gumeni, I.P. Trougakos, Cross talk of proteostasis and mitostasis in cellular homeodynamics, ageing, and disease, Oxidative Med. Cell. Longev. 2016 (2016) 4587691. [22] R.W. Goehe, X. Di, K. Sharma, et al., The autophagy-senescence connection in chemotherapy: must tumor cells (self) eat before they sleep? J. Pharmacol. Exp. Ther. 343 (3) (Dec 2012) 763–778. [23] Y. Zheng, Y. Lei, C. Hu, C. Hu, p53 regulates autophagic activity in senescent rat mesenchymal stromal cells, Exp. Gerontol. 75 (Mar 2016) 64–71. [24] D.P. Narendra, S.M. Jin, A. Tanaka, et al., PINK1 is selectively stabilized on impaired mitochondria to activate Parkin, PLoS Biol. 8 (1) (Jan 2010), e1000298. [25] N. Matsuda, S. Sato, K. Shiba, et al., PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy, J. Cell Biol. 189 (2) (Apr 19 2010) 211–221. [26] Y. Kim, J. Park, S. Kim, et al., PINK1 controls mitochondrial localization of Parkin through direct phosphorylation, Biochem. Biophys. Res. Commun. 377 (3) (Dec 19 2008) 975–980. [27] Y. He, J. Zhu, Y. Huang, H. Gao, Y. Zhao, Advanced glycation end product (AGE)-induced hepatic stellate cell activation via autophagy contributes to hepatitis Crelated fibrosis, Acta Diabetol. 52 (5) (Oct 2015) 959–969. [28] X. Hou, Z. Hu, H. Xu, et al., Advanced glycation endproducts trigger autophagy in cadiomyocyte via RAGE/PI3K/AKT/mTOR pathway, Cardiovasc. Diabetol. 13 (1) (2014) 78. [29] P. Hu, D. Lai, P. Lu, J. Gao, H. He, ERK and Akt signaling pathways are involved in advanced glycation end product-induced autophagy in rat vascular smooth muscle cells, Int. J. Mol. Med. 29 (4) (Apr 2012) 613–618. [30] Y. Maejima, S. Adachi, H. Ito, K. Hirao, M. Isobe, Induction of premature senescence in cardiomyocytes by doxorubicin as a novel mechanism of myocardial damage, Aging Cell 7 (2) (Mar 2008) 125–136. [31] S.D. Chen, T.K. Lin, D.I. Yang, et al., Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury, Biochem. Biophys. Res. Commun. 460 (2) (May 1 2015) 397–403. [32] X. Wei, Y. Qi, X. Zhang, et al., Cadmium induces mitophagy through ROS-mediated PINK1/Parkin pathway, Toxicol. Mech. Methods 24 (7) (2014) 504–511. [33] X. Wei, Y. Qi, X. Zhang, et al., ROS act as an upstream signal to mediate cadmiuminduced mitophagy in mouse brain, Neurotoxicology 46 (2015) 19–24. [34] J.W. Hartog, A.A. Voors, S.J. Bakker, A.J. Smit, D.J. van Veldhuisen, Advanced glycation end-products (AGEs) and heart failure: pathophysiology and clinical implications, Eur. J. Heart Fail. 9 (12) (Dec 2007) 1146–1155. [35] S. Willemsen, J.W. Hartog, M.R. Heiner-Fokkema, D.J. van Veldhuisen, A.A. Voors, Advanced glycation end-products, a pathophysiological pathway in the cardiorenal syndrome, Heart Fail. Rev. 17 (2) (Mar 2012) 221–228. [36] J. Nozynski, M. Zakliczynski, D. Konecka-Mrowka, et al., Advanced glycation end products and lipofuscin deposits share the same location in cardiocytes of the failing heart, Exp. Gerontol. 48 (2) (Feb 2013) 223–228. [37] E.D. Schleicher, E. Wagner, A.G. Nerlich, Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging, J. Clin. Invest. 99 (3) (Feb 1 1997) 457–468. [38] V.M. Monnier, G.T. Mustata, K.L. Biemel, et al., Cross-linking of the extracellular matrix by the maillard reaction in aging and diabetes: an update on “a puzzle nearing resolution”, Ann. N. Y. Acad. Sci. 1043 (Jun 2005) 533–544.
[39] N. Sakata, J. Meng, S. Jimi, M. Segawa, S. Takebayashi, Aging of aorta and atherosclerosis—role of nonenzymatic glycation of collagen, Nihon Ronen Igakkai Zasshi 32 (5) (May 1995) 336–343. [40] D. Badenhorst, M. Maseko, O.J. Tsotetsi, et al., Cross-linking influences the impact of quantitative changes in myocardial collagen on cardiac stiffness and remodelling in hypertension in rats, Cardiovasc. Res. 57 (3) (Mar 2003) 632–641. [41] J. Liu, J. Wang, X. Chen, C. Guo, Y. Guo, H. Wang, Ginkgo biloba extract EGB761 protects against aging-associated diastolic dysfunction in cardiomyocytes of D-galactose-induced aging rat, Oxidative Med. Cell. Longev. 2012 (2012) 418748. [42] D.J. Kurz, S. Decary, Y. Hong, J.D. Erusalimsky, Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells, J. Cell Sci. 113 (Pt 20) (Oct 2000) 3613–3622. [43] S.W. Lowe, C.J. Sherr, Tumor suppression by Ink4a-Arf: progress and puzzles, Curr. Opin. Genet. Dev. 13 (1) (Feb 2003) 77–83. [44] C. Tikellis, M.C. Thomas, B.E. Harcourt, et al., Cardiac inflammation associated with a Western diet is mediated via activation of RAGE by AGEs, Am. J. Physiol. Endocrinol. Metab. 295 (2) (Aug 2008) E323–E330. [45] D. Yan, X. Luo, Y. Li, et al., Effects of advanced glycation end products on calcium handling in cardiomyocytes, Cardiology 129 (2) (2014) 75–83. [46] R.E. Jimenez, D.A. Kubli, A.B. Gustafsson, Autophagy and mitophagy in the myocardium: therapeutic potential and concerns, Br. J. Pharmacol. 171 (8) (Apr 2014) 1907–1916. [47] B.C. Hammerling, A.B. Gustafsson, Mitochondrial quality control in the myocardium: cooperation between protein degradation and mitophagy, J. Mol. Cell. Cardiol. 75 (Oct 2014) 122–130. [48] R. Gómez-Sánchez, S.M.S. Yakhine-Diop, M. Rodríguez-Arribas, et al., mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines, Data Brief 7 (2016) 641–647. [49] S. Patschan, J. Chen, A. Polotskaia, et al., Lipid mediators of autophagy in stressinduced premature senescence of endothelial cells, Am. J. Physiol. Heart Circ. Physiol. 294 (3) (Mar 2008) H1119–H1129. [50] L. Li, Y.Q. Zhu, L. Jiang, W. Peng, Increased autophagic activity in senescent human dental pulp cells, Int. Endod. J. 45 (12) (Dec 2012) 1074–1079. [51] I. Kim, S. Rodriguez-Enriquez, J.J. Lemasters, Selective degradation of mitochondria by mitophagy, Arch. Biochem. Biophys. 462 (2) (Jun 15 2007) 245–253. [52] S. Rodriguez-Enriquez, I. Kim, R.T. Currin, J.J. Lemasters, Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes, Autophagy 2 (1) (Jan-Mar 2006) 39–46. [53] Y. Zheng, C.-J. Hu, R.-H. Zhuo, Y.-S. Lei, N.-N. Han, H. L, Inhibition of autophagy alleviates the senescent state of rat mesenchymal stem cells during long-term culture, Mol. Med. Rep. 10 (6) (2014 Dec) 3003–3008. [54] T. Hara, K. Nakamura, M. Matsui, et al., Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice, Nature 441 (7095) (Jun 15 2006) 885–889. [55] R. Mathew, V. Karantza-Wadsworth, E. White, Role of autophagy in cancer, Nat. Rev. Cancer 7 (12) (Dec 2007) 961–967. [56] T. Shintani, D.J. Klionsky, Autophagy in health and disease: a double-edged sword, Science (New York, N.Y.) 306 (5698) (Nov 5 2004) 990–995. [57] B. Levine, J. Yuan, Autophagy in cell death: an innocent convict? J. Clin. Invest. 115 (10) (Oct 2005) 2679–2688. [58] S. Sciarretta, P. Zhai, M. Volpe, J. Sadoshima, Pharmacological modulation of autophagy during cardiac stress, J. Cardiovasc. Pharmacol. 60 (3) (Sep 2012) 235–241. [59] A.R. Young, M. Narita, Connecting autophagy to senescence in pathophysiology, Curr. Opin. Cell Biol. 22 (2) (Apr 2010) 234–240. [60] K. Nishida, K. Otsu, Cell death in heart failure, Circ. J. 72 (Suppl. A) (2008) A17–A21. [61] F. Platini, R. Perez-Tomas, S. Ambrosio, L. Tessitore, Understanding autophagy in cell death control, Curr. Pharm. Des. 16 (1) (Jan 2010) 101–113. [62] J. Yang, Y. Zhou, X. Cheng, et al., Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells, Sci. Rep. 5 (2015) 7697. [63] B.Y. Law, W.K. Chan, S.W. Xu, et al., Natural small-molecule enhancers of autophagy induce autophagic cell death in apoptosis-defective cells, Sci. Rep. 4 (2014) 5510. [64] P. Sansanwal, B. Yen, W.A. Gahl, et al., Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis, J. Am. Soc. Nephrol. 21 (2) (Feb 2010) 272–283.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging Zhimin Zha a,1, Junhong Wang a,b,1, Xiangming Wang a, Miao Lu a, Yan Guo a,c,⁎ a b c
Department of Gerontology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of Cardiology, Shengze Hospital of Jiangsu Province, Suzhou, China
a r t i c l e
i n f o
Article history: Received 4 September 2016 Accepted 6 November 2016 Available online xxxx Keywords: Advanced glycation end products Cardiac senescence Mitophagy Receptor for advanced glycation end products PINK1
a b s t r a c t Context and objectives: Advanced glycation end products (AGEs) can induce senescence in cardiomyocytes. However, its underlying molecular mechanisms remain unknown. Methods: Neonatal rat cardiomyocytes were incubated with AGEs, and cellular senescence was evaluated by senescence-associated beta-galactosidase (SA-β-gal) activity and aging-associated p16 expression. In addition, mitophagic activity was evaluated by measuring the expression of the PINK1, Parkin, LC3 and p62 proteins. The mitophagy inhibitor cyclosporine A (CsA) or PINK1 siRNAs was then administered to cardiomyocytes to study the role of mitophagy in AGE-induced aging. Results: A significantly increased number of SA-β-gal positive cells and increased p16 protein levels were observed in cardiomyocytes treated with AGEs. Moreover, AGEs significantly increased the protein levels of PINK1 and Parkin as well as the LC3-II/LC3-I ratio, which occurred in a dose-dependent manner. However, the expression of p62 decreased significantly in the AGE group compared to the control. Surprisingly, both CsA and the knockdown of PINK1 by small-interfering RNA (siRNA) significantly decreased the LC3-II/LC3-I ratio and the PINK1 and Parkin protein levels in AGE-treated cardiomyocytes. Moreover, CsA treatment or knockdown of PINK1 expression attenuated the increased number of SA-β-gal positive cells and the upregulated p16 level in cardiomyocytes induced by AGEs. Conclusions: PINK1/Parkin-mediated mitophagy is involved in the process of cardiomyocyte senescence induced by AGEs, and a reduction in mitophagic activity might be a promising approach to block the senescent state in cardiomyocytes. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Aging, which is a process driven by randomly occurring damage and its accumulation, is an inevitable physiological phenomenon [1]. Ageassociated degenerative diseases such as cardiovascular diseases play a central role as the dominant cause of death due to the continuously increasing number of aged people. Cardiac aging is an independent risk factor for cardiovascular diseases and is associated with cardiac hypertrophy, diastolic dysfunction and compromised myocardial performance [2,3]. Many different hypotheses on the causes of aging have been proposed, including oxidative stress, mitochondrial dysfunction, telomere shortening, inflammation, and glycation, but the underlying mechanisms remain largely unknown.
⁎ Corresponding author at: Department of Gerontology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. E-mail address: [email protected] (Y. Guo). 1 These two authors contributed equally to this work.
Advanced glycation end products (AGEs) are a heterogeneous group of compounds that are formed by the non-enzymatic glycation of proteins, lipids and nucleic acids [1,4]. The accumulation of AGEs is one of the major mechanisms of aging and plays an important role in age-related diseases [5–8]. In the myocardium, the AGE-dependent modification of protein structure and related inhibition of function induce collagen cross-linking in the extracellular matrix and, subsequently, fibrotic cardiac remodeling, which leads to ventricular stiffness and diseases [9,10]. In addition, soluble AGEs can bind to specific cell surface receptors (RAGE being the best known) to activate several intracellular signaling pathways [11–13]. This is also associated with AGE-induced aging [12,14]. The aging process is coupled with another cell process, mitochondrial autophagy, which selectively targets and removes damaged or old mitochondria [15,16]. Mitophagy is reported to be an important process for establishing senescence [17,18]. Several studies have shown that changes in the mitophagy system are implicated in a plethora of agerelated diseases, such as Alzheimer's, Parkinson's and Huntington's diseases [19–21]. Recent observations have suggested that senescence
http://dx.doi.org/10.1016/j.ijcard.2016.11.161 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
2
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
and mitophagy share a number of common characteristics and are regulated by overlapping signaling pathways, which include the generation of reactive oxygen species, the induction of p53 and p21, and the dephosphorylation of retinoblastoma protein [22,23]. However, the potential roles of mitophagy during senescence remain unclear. The PINK1/Parkin pathway is a canonical mechanism that is involved in the regulation of mitophagy in mammal cells, and it was reported to be a mechanism in neurodegenerative disorders [24–26]. Further, accumulating evidence has indicated that AGEs can trigger mitophagy in a variety of cell types but that suppressing one of its receptors, RAGE, markedly reduces the formation of autolysosomes [27–29]. However, the effect of AGEs-activated mitophagy on AGEsinduced aging and the manner in which the mitophagic pathway changes are rarely reported in cardiomyocytes. For these reasons, we investigated the role of PINK1/Parkin-mediated mitophagy in AGEinduced senescence in cardiomyocytes. 2. Materials and methods 2.1. Drugs and reagents AGE-modified bovine serum albumin (AGE-BSA) was purchased from Calbiochem (Temecula, CA, USA). A neutralizing anti-RAGE antibody was purchased from R&D systems (Minneapolis, MN, USA). Cyclosporin A (CsA) was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies against PINK1, Parkin, p62 and p16 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-LC3 antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated goat antirabbit antibody was purchased from Abcam (Cambridge, MA, USA). The senescence-βGalactosidase staining kit was from the Beyotime Institute of Biotechnology (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were from Grand Island Biological Company (Gibco, Grand Island, NY, USA). The protein quantification assay kit was purchased from Bio-Rad (Hercules, CA, USA). 2.2. Cell culture and treatment The animal handling and use protocols complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No.85-23, revised 1996) and were approved by the Animal Care and Use Committee of Nanjing Medical University. Ventricular myocytes were isolated from neonatal SpragueDawley rats (1 to 3 days old) as described previously [30]. Hearts were removed quickly and washed three times with cold phosphate buffered saline (PBS). The left heart tissues were minced with scissors into 1 mm3 fragments and were incubated in collagenase II solutions for further digestion. The homogenized tissue was allowed to adhere to the 100mm dish in culture media (DMEM) containing 10% FBS for 2 h at 37 °C. Fibroblasts were separated by adhesion onto the dish during this process. The non-adherent cells, neonatal rat ventricular cardiomyocytes, were plated in a culture plate and cultured in DMEM medium containing 10% (v/v) FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The medium was replaced every 48 h. After 3 days of culture, when the cells reached 80% confluence, the cardiomyocytes could be used in the subsequent experiments. Cardiomyocytes were treated with different concentrations of AGEs (50, 100, 200 μg/ml) for 48 h to investigate the effects of AGEs on cardiomyocytes. Then, the cardiomyocytes were incubated with AGEs (100 μg/ml), RAGE antibody (2 μg/ml) or CsA (0.5 μmol/l), as specifically indicated, and cultured for 48 h. 2.3. Beta-galactosidase activity Senescence-associated beta-galactosidase (SA-β-gal) activity was measured using the β-gal staining kit at pH 6.0 following the manufacturer's protocol (Beyotime Institute of Biotechnology, Wuhan, China). Briefly, cells were seeded into six-well plates, cultured with the reagents as mentioned above for 48 h, fixed for 15 min at room temperature with 1 mL of fixative solution and then washed three times with phosphate-buffered saline. Next, the cells were incubated overnight at 37 °C with a staining solution mix containing X-gal. After being rinsed with PBS, cells were observed for the development of the blue coloration with a microscope at a magnification of ×400. 2.4. siRNA interference and gene transfection The siRNA oligonucleotides targeting PINK1 were proven to be effective by other researchers [31] and synthesized by GenePharma (GenePharma, Shanghai, China). The sequences were as follows: negative control siRNA (NC): 5´-GAU CAU ACG UGC GAU CAG ATT-3′ and PINK1 siRNA: 5′ -GCU GCA AUG CCG CUG UGU ATT-3′ . siRNAs were then transfected into cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Lipofectamine 3000 and siRNA were diluted in Opti-MEM medium. After briefly vortexing, the mixture was incubated at room temperature for 10– 15 min to allow the formation of siRNA-Lipofectamine 3000 complexes and then added to the cells in DMEM medium containing serum/antibiotic. After incubation for 48 h, the cells were used for the following experiments.
2.5. Western blot analysis For western blot, cells were lysed with ice-cold Cell Lysis Reagent containing protease and phosphatase inhibitors (BiYun Tian, China, P0013B) to extract total proteins. After the protein samples were heat-denatured, equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes by electrophoresis. After transfer, the membranes were blocked with 5% non-fat milk and incubated overnight with primary antibodies against PINK1, Parkin, p62, LC3 and p16. Membranes were subsequently washed in Tris-buffered saline plus Tween-20 (TBST) and incubated with anti-rabbit HRP-conjugated secondary antibodies for two hours at 4 °C. After washing the membrane in TBST three times, the protein levels were detected by chemiluminescence using an enhanced chemiluminescence (ECL) detection kit (Millipore Corporation, Temecula, CA, USA). The level of GAPDH was used as an internal control. 2.6. Statistical analysis All data were obtained from at least 3 independent experiments. The results were presented as the mean ± SD. Statistical analyses were carried out using the GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). One-way ANOVA was used to compare the difference in those groups. Statistical significance was determined at a P value of b0.05.
3. Results 3.1. AGEs induces cellular senescence in neonatal rat cardiomyocytes To investigate whether cardiomyocytes treated with AGEs showed a senescent phenotype, neonatal rat cardiomyocytes were treated with different concentrations of AGEs. The frequency of SA-β-gal-positive staining and the expression level of the senescence marker, p16, were investigated to evaluate the senescence level of the cell population. As shown in Figs. 3 and 5, 48 h of incubation with AGEs resulted in significantincreases in both the cellular SA-β-gal activity and p16 levels compared to the control group, suggesting that AGEs can directly induce senescence in cardiomyocytes. 3.2. Increased mitophagy in AGEs-treated cardiomyocytes To evaluate mitophagic activity in AGE-induced senescent cardiomyocytes, mitophagy was investigated by analyzing the levels of mitophagy-related proteins. The conversion of cellular protein LC3-I to LC3-II and the level of p62 are two critical hallmarks of the mitophagy process. As shown in Fig. 1A and B, an accumulation of LC3-II and a decreased level of P62 were observed in AGEs group compared to the control, indicating elevated mitophagic activity in AGE-induced senescent cardiomyocytes. Furthermore, this increase occurred in a dosedependent manner with increasing concentration of AGEs, and the peak value was observed when the concentration of AGEs was 200 μg/ml. Two other important mitophagy-associated proteins, PINK1 and Parkin, were also investigated by western blotting. Senescent cardiomyocytes exhibited significantly increased expression levels of PINK1 and Parkin compared to the control group (Fig. 1C and D). These results suggest that AGE-induced senescent cardiomyocytes have high mitophagic activity. 3.3. RAGE-mediated, AGEs-induced senescence and mitophagy in cardiomyocytes In the AGEs group, the percentage of SA-β-gal-positive cardiomyocytes and the expression of p16 were significantly higher than those in the normal control group. However, the RAGE antibody significantly decreased both the SA-β-gal activity and p16 level in the AGEs group (Fig. 3A and B). After cardiomyocytes were pretreated with anti-RAGE for 2 h, the LC3-II/LC3-I ratio and the PINK1, Parkin and p62 levels were determined by western blot. Significant decreases in the LC3-II/LC3-I ratio and in the PINK1 and Parkin levels were observed in comparison to the AGEs group. In addition, the expression of p62 was increased significantly in the AGEs + RAGE antibody group (Fig. 2A-D).
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
3.4. Inhibition of mitophagy alleviates senescence in AGE-treated cardiomyocytes To study the role of mitophagy in AGE-induced cardiomyocyte senescence, the cardiomyocytes were incubated with a mitophagy inhibitor (0.5 μmol/l CsA) [32,33] and AGEs. Our results revealed that the LC3-II/LC3-I ratio was significantly reduced and that the p62 level was markedly increased when CsA was administered to the AGEinduced senescent cardiomyocytes for two days. (Fig. 2A and B). These
3
data may indicate that mitophagy was involved in the process of AGEinduced cardiomyocyte senescence. In addition, the senescence status of CsA treated cardiomyocytes was further analyzed. The rate of SA-β-gal-positive staining was significantly reduced after the cells were coincubated with CsA (Fig. 3A). Consistent with effect on SA-β-gal, the expression level of p16 protein was also reduced in the CsA group (Fig. 3B). This indicated that mitophagy is involved in the process of AGE-induced cardiomyocytes senescence.
Fig. 1. AGEs induce mitophagy in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to increasing concentrations of AGEs (0–200 μg/ml) for 48 h. LC3 (A), p62 (B), PINK1 (C) and Parkin (D) levels were measured by western blotting. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
4
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
3.5. Mitophagy-accelerated senescence in cardiomyocytes is dependent on PINK1/Parkin activation Because the PINK1/Parkin signaling pathway plays a pivotal role in mitophagy, the protein levels of PINK1 and Parkin were analyzed in AGE-induced senescent cardiomyocytes. Our results demonstrated
that the PINK1 and Parkin levels were significantly increased compared to those of the control group (Fig. 2C and D). Next, the gene silencing of PINK1 was used to study the role of the PINK/Parkin pathway in AGEinduced senescent cardiomyocytes. Our results showed that the decreased PINK1 expression in PINK1 siRNA group significantly decreased the LC3-II/LC3-I ratio compared to the AGEs group (Fig. 4A and B).
Fig. 2. RAGE antibody and CsA both inhibit AGEs-induced mitophagy in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to 100 μg/ml AGEs alone or in combination with 2 μg/ ml RAGE antibody or 0.5 μmol/L CsA. LC3 (A), p62 (B), PINK1 (C) and Parkin (D) levels were measured by western blotting. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control. $$ P b 0.01 compared with the corresponding AGEs treatment alone.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
Consequently, the senescence status of the AGEs-treated cardiomyocytes was further studied in the cells that were coincubated with the PINK1 siRNA. Significant decreases in the percentage of SA-βgal-positive cells and the expression level of the p16 protein were observed in the PINK1 siRNA-treated group (PINK1 siRNA + AGEs) compared to cells in the control AGEs group (Fig. 5A and B). Collectively, our data revealed that PINK1/Parkin pathway-mediated mitophagy might play a pivotal role in AGE-induced cardiomyocyte senescence. 4. Discussion AGEs are responsible for degenerative diseases such as cardiovascular diseases due to their accumulation within molecules, cells and tissues during aging [34–37]. Glycation modifies the structure and function of proteins and induces an increase in stiffness and a reduction in elasticity in vessels and heart via crosslinking in the extracellular matrix [38–40], which suggests that AGEs are closely associated with cardiac aging. Our previous study demonstrated that D-galactose, a reducing
5
sugar that is involved in the formation of AGEs, could induce cardiomyocyte senescence [41]. In our study, AGEs increased both the cellular SAβ-gal activity and the p16 level, which are the most widely used biomarkers for cardiomyocyte senescence. SA-β-gal is an early marker of cellular senescence because it reflects the function of the lysosomes and appears as the result of their increased biogenesis [42]. The p16 protein, a cyclin-dependent kinase (CDK) 4/6 inhibitor [43], functions in the restriction of G1/S cell cycle progression and in the induction of senescence. Consequently, our results may indicate that AGEs play an important role in the process of cardiac aging. In addition, RAGE, a specific receptor of AGEs, is expressed in various cell types, including cardiomyocytes. The binding of AGEs with RAGE receptors could influence cardiac function by activating multiple processes, including inflammatory responses, oxidative stress, cardiac fibrosis and impaired calcium metabolism in the cardiovascular system [4,44,45]. In the present study, the inhibition of RAGE with a RAGE-antibody reduced both the SA-β-gal activity and p16 level during AGE exposure, indicating that RAGE mediated the AGE-elicited senescence of cardiomyocytes.
Fig. 3. RAGE antibody and CsA attenuate AGEs-induced senescence in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to 100 μg/ml AGEs alone or in combination with 2 μg/ml RAGE antibody or 0.5 μmol/l CsA. (A) SA-β-gal assay. Representative image of SA-β-gal positive cells (×400). SA-β-gal positive cells are stained in blue. (B) The expression level of p16 was determined by western blot. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control. $$ P b 0.01 compared with the corresponding AGEs treatment alone. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
6
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
Fig. 4. Downregulation of PINK1 attenuates the mitophagy mediated by AGEs in cardiomyocytes. Neonatal rat cardiomyocytes were exposed to 100 μg/ml AGEs after transfection with negative control siRNA or PINK1 siRNA. PINK1 (A) and LC3 (B) levels were measured by western blotting. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control. $$ P b 0.01 compared with the corresponding AGEs treatment alone.
These results are consistent with previous studies [4,14]. Together, these data demonstrated that the AGEs-RAGE axis plays an important role in the process of cardiomyocyte senescence. Mitophagy plays a crucial role in maintaining cardiac homeostasis [46,47]. There are multiple stages, structures, and proteins involved in this process [16]. In our study, we found that the LC3-II/LC3-I ratio was upregulated and that the level of p62 was downregulated during the process of AGE-induced senescence in cardiomyocytes. An increase in LC3-II protein is considered a marker for elevated autophagosome formation, and a decrease in p62 protein is a marker for increased autophagic turnover [48]. Accordingly, our results indicated that mitophagy might be involved in the cardiomyocyte senescence induced by AGEs. Mitophagy and senescence are considered to be two distinct cellular events; however, changes in the expression of mitophagic markers have been observed in different senescent cell types, which indicates that these two events are closely related [49,50]. Our findings showed that inhibiting mitophagy activation via a mitophagy inhibitor, CsA [51,52], contributed to the reduced SA-β-gal activity and reduced p16 expression in AGEs-treated cardiomyocytes. These results indicated that mitophagy might facilitate the development of cardiac aging induced by AGEs. These results also show that inhibition of mitophagy alleviates the AGE-induced senescent state in cardiomyocytes, which is in line with previous data on other cell types [17,22,49,53]. However, a number of studies support an inverse association between mitophagy and senescence, showing that mitophagic activity decreased during aging [15,54,55]. It is as though mitophagy plays a Janus-type role in that they can be either cytoprotective or cytotoxic [56–58]. This discrepancy may result from differences in the experimental and clinical conditions (different triggers, cellular and microenvironmental conditions) and the autophagic extent. It is possible that autophagy, under baseline conditions, is required to repair mild damage. In this context, autophagy is a homeostatic mechanism for maintaining cellular function and survival. However, in other circumstances, e.g., specific
disease settings, excess levels of autophagy facilitate the loss of cellular functions, including cell senescence [53,59]. There are also numerous pieces of evidence suggesting that autophagy is now considered a death execution mechanism (i.e., type II programmed cell death) [60–63]. The abnormal mitophagy observed in previous studies is consistent with this hypothesis [64]. Taken together, we assumed that excessive mitophagy might impair cardiac function, which could ultimately result in cardiac aging when the cardiomyocytes were exposed to AGEs. PINK1 and Parkin activation is a canonical mechanism for mitophagy regulation in most mammals [24–26]. Upon mitochondrial damage, PINK1 globally accumulates on depolarized mitochondria and recruits Parkin from the cytosol to the mitochondria, which is part of an ubiquitylation-related process for the selective removal of mitochondria. Thus, we hypothesized that the PINK1/Parkin pathway might be involved in regulating AGEs-induce cardiomyocyte senescence. Interestingly, our results demonstrated that the depletion of PINK1 in cardiomyocytes resulted in a remarkable attenuation both in mitophagy activity and in cardiomyocyte senescence induced by AGEs. These findings might indicate that PINK1/Parkin mediated-mitophagy may be involved in cardiac aging induced by AGEs. Therefore, our results may indicate that PINK1/Parkin mediated mitophagy plays an important role in AGEs-induced senescence of cardiomyocytes. The current study has some limitations. First, we only studied the PINK1/Parkin signaling pathway, which is not the only regulator of mitophagy. We did not look at other signal pathways that can regulate mitophagy in this study. More importantly, additional experiments on animals or humans should be done, and more work should be focused in the future on the specific effects of mitophagy on cardiac aging. However, taken together, the present study, for the first time, has revealed that AGEs-induced mitophagy promotes senescence in cardiomyocytes via RAGE and activation of the PINK1/Parkin pathway. Our current work provides a new insight into the understanding of mitophagy and aging
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
7
Fig. 5. PINK1 silencing alleviates the senescent state of cardiomyocytes treated with AGEs. Neonatal rat cardiomyocytes were exposed to 100 μg/ml AGEs after negative control siRNA or PINK1 siRNA transfection. (A) SA-β-gal assay. Representative micrographs depict morphology of SA-β-gal positive cells (×400). SA-β-gal positive cells are stained in blue. (B) The expression level of p16 was determined by western blot. Data are presented as the mean ± SD, n = 3 in each group. **P b 0.01 compared with control. $$ P b 0.01 compared with the corresponding AGEs treatment alone. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in cardiomyocytes. Moreover, these findings may provide a potential strategy for regulating mitophagy appropriately, that is, in a way that may counteract cardiac aging. Conflict of interest statement The authors declare no potential conflicts of interest were disclosed. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC 81570328, Wang Junhong) and the “Sixth-Peak Talent” of Jiangsu Province (2011WSN-029 to Prof. Guo Yan and 2013WSN-036 to Dr. Wang Junhong). Prof. Guo Yan was also support by the Health Department of Jiangsu Province (z201301). References [1] A. Simm, B. Muller, N. Nass, et al., Protein glycation - between tissue aging and protection, Exp. Gerontol. 68 (Aug 2015) 71–75. [2] R.A. Boon, K. Iekushi, S. Lechner, et al., MicroRNA-34a regulates cardiac ageing and function, Nature 495 (7439) (Mar 7 2013) 107–110.
[3] Y.A. Chiao, MicroRNA-34a: a new piece in the cardiac aging puzzle, Circ. Cardiovasc. Genet. 6 (4) (Aug 2013) 437–438. [4] M. Fang, J. Wang, S. Li, Y. Guo, Advanced glycation end-products accelerate the cardiac aging process through the receptor for advanced glycation end-products/ transforming growth factor-beta-Smad signaling pathway in cardiac fibroblasts, Geriatr. Gerontol. Int. 16 (4) (Apr 2016) 522–527. [5] A. Simm, Protein glycation during aging and in cardiovascular disease, J. Proteome 92 (Oct 30 2013) 248–259. [6] L. Robert, J. Labat-Robert, Role of the Maillard reaction in aging and age-related diseases. Studies at the cellular-molecular level, Clin. Chem. Lab. Med. 52 (1) (Jan 1 2014) 5–10. [7] M. Cardenas-Leon, E. Diaz-Diaz, R. Arguelles-Medina, P. Sanchez-Canales, V. DiazSanchez, F. Larrea, Glycation and protein crosslinking in the diabetes and ageing pathogenesis, Rev. Investig. Clin. 61 (6) (Nov-Dec 2009) 505–520. [8] N. Nass, B. Bartling, A. Navarrete Santos, et al., Advanced glycation end products, diabetes and ageing, Z. Gerontol. Geriatr. 40 (5) (Oct 2007) 349–356. [9] S.Y. Choi, H.J. Chang, S.I. Choi, et al., Long-term exercise training attenuates agerelated diastolic dysfunction: association of myocardial collagen cross-linking, J. Korean Med. Sci. 24 (1) (Feb 2009) 32–39. [10] S. Willemsen, J.W. Hartog, Y.M. Hummel, et al., Tissue advanced glycation end products are associated with diastolic function and aerobic exercise capacity in diabetic heart failure patients, Eur. J. Heart Fail. 13 (1) (Jan 2011) 76–82. [11] K.H. Gaens, C.D. Stehouwer, C.G. Schalkwijk, Advanced glycation endproducts and its receptor for advanced glycation endproducts in obesity, Curr. Opin. Lipidol. 24 (1) (Feb 2013) 4–11. [12] C. Ott, K. Jacobs, E. Haucke, A. Navarrete Santos, T. Grune, A. Simm, Role of advanced glycation end products in cellular signaling, Redox Biol. 2 (2014) 411–429.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161
8
Z. Zha et al. / International Journal of Cardiology xxx (2016) xxx–xxx
[13] A.L. Tan, J.M. Forbes, M.E. Cooper, AGE, RAGE, and ROS in diabetic nephropathy, Semin. Nephrol. 27 (2) (Mar 2007) 130–143. [14] J. Liu, K. Huang, G.Y. Cai, et al., Receptor for advanced glycation end-products promotes premature senescence of proximal tubular epithelial cells via activation of endoplasmic reticulum stress-dependent p21 signaling, Cell. Signal. 26 (1) (Jan 2014) 110–121. [15] J.J. Lemasters, Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging, Rejuvenation Res. 8 (1) (Spring 2005) 3–5. [16] A. Hamacher-Brady, N.R. Brady, Mitophagy programs: mechanisms and physiological implications of mitochondrial targeting by autophagy, Cell. Mol. Life Sci. 73 (4) (Feb 2016) 775–795. [17] M. Narita, A.R. Young, M. Narita, Autophagy facilitates oncogene-induced senescence, Autophagy 5 (7) (Oct 2009) 1046–1047. [18] A. Shaik, A. Schiavi, N. Ventura, Mitochondrial autophagy promotes healthy aging, Cell Cycle 15 (14) (2016 Jul 17) 1805–1806. [19] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (1) (Jan 11 2008) 27–42. [20] A. Perl, mTOR activation is a biomarker and a central pathway to autoimmune disorders, cancer, obesity, and aging, Ann. N. Y. Acad. Sci. 1346 (1) (Jun 2015) 33–44. [21] S. Gumeni, I.P. Trougakos, Cross talk of proteostasis and mitostasis in cellular homeodynamics, ageing, and disease, Oxidative Med. Cell. Longev. 2016 (2016) 4587691. [22] R.W. Goehe, X. Di, K. Sharma, et al., The autophagy-senescence connection in chemotherapy: must tumor cells (self) eat before they sleep? J. Pharmacol. Exp. Ther. 343 (3) (Dec 2012) 763–778. [23] Y. Zheng, Y. Lei, C. Hu, C. Hu, p53 regulates autophagic activity in senescent rat mesenchymal stromal cells, Exp. Gerontol. 75 (Mar 2016) 64–71. [24] D.P. Narendra, S.M. Jin, A. Tanaka, et al., PINK1 is selectively stabilized on impaired mitochondria to activate Parkin, PLoS Biol. 8 (1) (Jan 2010), e1000298. [25] N. Matsuda, S. Sato, K. Shiba, et al., PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy, J. Cell Biol. 189 (2) (Apr 19 2010) 211–221. [26] Y. Kim, J. Park, S. Kim, et al., PINK1 controls mitochondrial localization of Parkin through direct phosphorylation, Biochem. Biophys. Res. Commun. 377 (3) (Dec 19 2008) 975–980. [27] Y. He, J. Zhu, Y. Huang, H. Gao, Y. Zhao, Advanced glycation end product (AGE)-induced hepatic stellate cell activation via autophagy contributes to hepatitis Crelated fibrosis, Acta Diabetol. 52 (5) (Oct 2015) 959–969. [28] X. Hou, Z. Hu, H. Xu, et al., Advanced glycation endproducts trigger autophagy in cadiomyocyte via RAGE/PI3K/AKT/mTOR pathway, Cardiovasc. Diabetol. 13 (1) (2014) 78. [29] P. Hu, D. Lai, P. Lu, J. Gao, H. He, ERK and Akt signaling pathways are involved in advanced glycation end product-induced autophagy in rat vascular smooth muscle cells, Int. J. Mol. Med. 29 (4) (Apr 2012) 613–618. [30] Y. Maejima, S. Adachi, H. Ito, K. Hirao, M. Isobe, Induction of premature senescence in cardiomyocytes by doxorubicin as a novel mechanism of myocardial damage, Aging Cell 7 (2) (Mar 2008) 125–136. [31] S.D. Chen, T.K. Lin, D.I. Yang, et al., Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury, Biochem. Biophys. Res. Commun. 460 (2) (May 1 2015) 397–403. [32] X. Wei, Y. Qi, X. Zhang, et al., Cadmium induces mitophagy through ROS-mediated PINK1/Parkin pathway, Toxicol. Mech. Methods 24 (7) (2014) 504–511. [33] X. Wei, Y. Qi, X. Zhang, et al., ROS act as an upstream signal to mediate cadmiuminduced mitophagy in mouse brain, Neurotoxicology 46 (2015) 19–24. [34] J.W. Hartog, A.A. Voors, S.J. Bakker, A.J. Smit, D.J. van Veldhuisen, Advanced glycation end-products (AGEs) and heart failure: pathophysiology and clinical implications, Eur. J. Heart Fail. 9 (12) (Dec 2007) 1146–1155. [35] S. Willemsen, J.W. Hartog, M.R. Heiner-Fokkema, D.J. van Veldhuisen, A.A. Voors, Advanced glycation end-products, a pathophysiological pathway in the cardiorenal syndrome, Heart Fail. Rev. 17 (2) (Mar 2012) 221–228. [36] J. Nozynski, M. Zakliczynski, D. Konecka-Mrowka, et al., Advanced glycation end products and lipofuscin deposits share the same location in cardiocytes of the failing heart, Exp. Gerontol. 48 (2) (Feb 2013) 223–228. [37] E.D. Schleicher, E. Wagner, A.G. Nerlich, Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging, J. Clin. Invest. 99 (3) (Feb 1 1997) 457–468. [38] V.M. Monnier, G.T. Mustata, K.L. Biemel, et al., Cross-linking of the extracellular matrix by the maillard reaction in aging and diabetes: an update on “a puzzle nearing resolution”, Ann. N. Y. Acad. Sci. 1043 (Jun 2005) 533–544.
[39] N. Sakata, J. Meng, S. Jimi, M. Segawa, S. Takebayashi, Aging of aorta and atherosclerosis—role of nonenzymatic glycation of collagen, Nihon Ronen Igakkai Zasshi 32 (5) (May 1995) 336–343. [40] D. Badenhorst, M. Maseko, O.J. Tsotetsi, et al., Cross-linking influences the impact of quantitative changes in myocardial collagen on cardiac stiffness and remodelling in hypertension in rats, Cardiovasc. Res. 57 (3) (Mar 2003) 632–641. [41] J. Liu, J. Wang, X. Chen, C. Guo, Y. Guo, H. Wang, Ginkgo biloba extract EGB761 protects against aging-associated diastolic dysfunction in cardiomyocytes of D-galactose-induced aging rat, Oxidative Med. Cell. Longev. 2012 (2012) 418748. [42] D.J. Kurz, S. Decary, Y. Hong, J.D. Erusalimsky, Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells, J. Cell Sci. 113 (Pt 20) (Oct 2000) 3613–3622. [43] S.W. Lowe, C.J. Sherr, Tumor suppression by Ink4a-Arf: progress and puzzles, Curr. Opin. Genet. Dev. 13 (1) (Feb 2003) 77–83. [44] C. Tikellis, M.C. Thomas, B.E. Harcourt, et al., Cardiac inflammation associated with a Western diet is mediated via activation of RAGE by AGEs, Am. J. Physiol. Endocrinol. Metab. 295 (2) (Aug 2008) E323–E330. [45] D. Yan, X. Luo, Y. Li, et al., Effects of advanced glycation end products on calcium handling in cardiomyocytes, Cardiology 129 (2) (2014) 75–83. [46] R.E. Jimenez, D.A. Kubli, A.B. Gustafsson, Autophagy and mitophagy in the myocardium: therapeutic potential and concerns, Br. J. Pharmacol. 171 (8) (Apr 2014) 1907–1916. [47] B.C. Hammerling, A.B. Gustafsson, Mitochondrial quality control in the myocardium: cooperation between protein degradation and mitophagy, J. Mol. Cell. Cardiol. 75 (Oct 2014) 122–130. [48] R. Gómez-Sánchez, S.M.S. Yakhine-Diop, M. Rodríguez-Arribas, et al., mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines, Data Brief 7 (2016) 641–647. [49] S. Patschan, J. Chen, A. Polotskaia, et al., Lipid mediators of autophagy in stressinduced premature senescence of endothelial cells, Am. J. Physiol. Heart Circ. Physiol. 294 (3) (Mar 2008) H1119–H1129. [50] L. Li, Y.Q. Zhu, L. Jiang, W. Peng, Increased autophagic activity in senescent human dental pulp cells, Int. Endod. J. 45 (12) (Dec 2012) 1074–1079. [51] I. Kim, S. Rodriguez-Enriquez, J.J. Lemasters, Selective degradation of mitochondria by mitophagy, Arch. Biochem. Biophys. 462 (2) (Jun 15 2007) 245–253. [52] S. Rodriguez-Enriquez, I. Kim, R.T. Currin, J.J. Lemasters, Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes, Autophagy 2 (1) (Jan-Mar 2006) 39–46. [53] Y. Zheng, C.-J. Hu, R.-H. Zhuo, Y.-S. Lei, N.-N. Han, H. L, Inhibition of autophagy alleviates the senescent state of rat mesenchymal stem cells during long-term culture, Mol. Med. Rep. 10 (6) (2014 Dec) 3003–3008. [54] T. Hara, K. Nakamura, M. Matsui, et al., Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice, Nature 441 (7095) (Jun 15 2006) 885–889. [55] R. Mathew, V. Karantza-Wadsworth, E. White, Role of autophagy in cancer, Nat. Rev. Cancer 7 (12) (Dec 2007) 961–967. [56] T. Shintani, D.J. Klionsky, Autophagy in health and disease: a double-edged sword, Science (New York, N.Y.) 306 (5698) (Nov 5 2004) 990–995. [57] B. Levine, J. Yuan, Autophagy in cell death: an innocent convict? J. Clin. Invest. 115 (10) (Oct 2005) 2679–2688. [58] S. Sciarretta, P. Zhai, M. Volpe, J. Sadoshima, Pharmacological modulation of autophagy during cardiac stress, J. Cardiovasc. Pharmacol. 60 (3) (Sep 2012) 235–241. [59] A.R. Young, M. Narita, Connecting autophagy to senescence in pathophysiology, Curr. Opin. Cell Biol. 22 (2) (Apr 2010) 234–240. [60] K. Nishida, K. Otsu, Cell death in heart failure, Circ. J. 72 (Suppl. A) (2008) A17–A21. [61] F. Platini, R. Perez-Tomas, S. Ambrosio, L. Tessitore, Understanding autophagy in cell death control, Curr. Pharm. Des. 16 (1) (Jan 2010) 101–113. [62] J. Yang, Y. Zhou, X. Cheng, et al., Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells, Sci. Rep. 5 (2015) 7697. [63] B.Y. Law, W.K. Chan, S.W. Xu, et al., Natural small-molecule enhancers of autophagy induce autophagic cell death in apoptosis-defective cells, Sci. Rep. 4 (2014) 5510. [64] P. Sansanwal, B. Yen, W.A. Gahl, et al., Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis, J. Am. Soc. Nephrol. 21 (2) (Feb 2010) 272–283.
Please cite this article as: Z. Zha, et al., Involvement of PINK1/Parkin-mediated mitophagy in AGE-induced cardiomyocyte aging, Int J Cardiol (2016), http://dx.doi.org/10.1016/j.ijcard.2016.11.161